From the Centro de Investigaciones Cardiovasculares, Facultad de Ciencias Médicas, Universidad Nacional de La Plata, 60 y 120, 1900 La Plata, Argentina
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ABSTRACT |
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Previous experiments have shown that acidosis enhances isoproterenol-induced phospholamban (PHL) phosphorylation (Mundiña-Weilenmann, C., Vittone, L., Cingolani, H. E., Orchard, C. H. (1996) Am. J. Physiol. 270, C107-C114). In the present experiments, performed in isolated Langendorff perfused rat hearts, phosphorylation site-specific antibodies to PHL combined with the quantitative measurement of 32P incorporation into PHL were used as experimental tools to gain further insight into the mechanism involved in this effect. At all isoproterenol concentrations tested (3-300 nM), phosphorylation of Thr17 of PHL was significantly higher at pHo 6.80 than at pHo 7.40, without significant changes in Ser16 phosphorylation. This increase in Thr17 phosphorylation was associated with an enhancement of the isoproterenol-induced relaxant effect. In the absence of isoproterenol, the increase in [Ca]o at pHo 6.80 (but not at pHo 7.40) evoked an increase in PHL phosphorylation that was exclusively due to an increase in Thr17 phosphorylation and that was also associated with a significant relaxant effect. This effect and the phosphorylation of Thr17 evoked by acidosis were both offset by the Ca2+/calmodulin-dependent protein kinase II inhibitor KN-62. In the presence of isoproterenol, either the increase in [Ca]o or the addition of a 1 µM concentration of the phosphatase inhibitor okadaic acid was able to mimic the increase in isoproterenol-induced Thr17 phosphorylation produced by acidosis. In contrast, these two interventions have opposite effects on phosphorylation of Ser16. Whereas the increase in [Ca]o significantly decreased phosphorylation of Ser16, the addition of okadaic acid significantly increased the phosphorylation of this residue. The results are consistent with the hypothesis that the increase in phospholamban phosphorylation produced by acidosis in the presence of isoproterenol is the consequence of two different mechanisms triggered by acidosis: an increase in [Ca2+]i and an inhibition of phosphatases.
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INTRODUCTION |
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The sarcoplasmic reticulum
(SR)1 Ca2+-ATPase
plays a pivotal role in the contraction and relaxation process of
myocardial cells (1). Ca2+-ATPase activity is tonically
inhibited by the interaction with another SR protein named
phospholamban. In vitro experiments indicate that
phosphorylation of phospholamban occurs by either
cAMP-dependent protein kinase (PKA) at Ser16 or
Ca2+/calmodulin-dependent protein kinase II
(CaMKII) at Thr17 (2). Phosphorylation of phospholamban
causes dissociation of this protein from the pump, thus increasing
ATPase activity and the rate of Ca2+ uptake by the SR (3).
In the intact beating heart, -adrenoreceptor stimulation
phosphorylates phospholamban at both Ser16 and
Thr17 (4, 5). Experimental evidence from our own laboratory
strongly suggested that this dual phosphorylation requires both
stimulation of the PKA and CaMKII cascades of phospholamban
phosphorylation and simultaneous inhibition of phospholamban
phosphatase (5). These two prerequisites appear to be fulfilled by
-adrenoreceptor stimulation, which, as a result of PKA activation,
triggers the activation of CaMKII by increasing intracellular
Ca2+ and produces the inhibition of PP1, the major
phosphatase that dephosphorylates phospholamban (6-11). This may be
the reason why several attempts to phosphorylate phospholamban by
increasing [Ca2+]i through cAMP-independent
mechanisms have failed (12-15). Inhibition of phospholamban
phosphatase was required (5).
The effect of -adrenoreceptor stimulation on phospholamban
phosphorylation and myocardial relaxation is dependent on the acid-base
status of the myocardium. It has been shown that acidosis enhances
isoproterenol-induced phospholamban phosphorylation and myocardial
relaxation (16). The mechanism of this action remains unknown. Among
several different effects, acidosis increases intracellular calcium
levels (17, 18) and inhibits PP1 activity (16) in the rat myocardium.
Thus, it seems reasonable to consider that the acidosis enhancement of
the isoproterenol-induced increase in phospholamban phosphorylation
could be due to a further increase either in Ser16
phosphorylation (by inhibition of PP1) and/or in Thr17
phosphorylation (by inhibition of PP1 and/or activation of CaMKII).
In this work, immunodetection of site-specific phosphorylated phospholamban was used in combination with the classical isotopic labeling technique of quantification of phospholamban phosphorylation to investigate the contribution of each phosphorylation site of phospholamban to the increase in the isoproterenol-induced phosphorylation of this protein evoked by acidosis. The possible mechanisms involved in this effect were also explored. Simultaneous measurements of mechanical parameters will provide a clue to the functional consequences of the effects observed.
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EXPERIMENTAL PROCEDURES |
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Heart Perfusions--
Experiments were performed in isolated
hearts from male Wistar rats (250-350 g of body weight) perfused
according to the Langendorff technique as described previously (13).
The composition of the physiological salt solution (PSS) was 128.3 mM NaCl, 4.7 mM KCl, 1.35 mM
CaCl2, 20.2 mM NaHCO3, 0.4 mM NaH2PO4, 1.1 mM
MgCl2, 11.1 mM glucose, and 0.04 mM
Na2EDTA; this solution was equilibrated with 95%
O2 and 5% CO2 to give an extracellular pH
(pHo) of 7.40 ± 0.01 or with 80% O2 and 20%
CO2 (pHo 6.80 ± 0.02) in the experiments of
hypercapnic acidosis. The mechanical activity of the heart was assessed
by passing into the left ventricle a latex balloon connected to a
pressure transducer (Namic, Perceptor DT disposable transducer). The
balloon was filled with aqueous solution to achieve a left ventricular
end diastolic pressure of 8-14 mm Hg. Isovolumic pressure and its
first derivative were recorded on a four-channel pen recorder (Gould
Model RS 3400) fitted with a transducer amplifier (Gould Model
13-4615-50) and a differentiating amplifier (Gould Model 13-4615-71).
Hearts were perfused with PSS at pHo 7.40 for 10-15 min for
stabilization and then for the next 3 min with either PSS (control) or
different interventions as described under "Results." To quantify
32P incorporation into phospholamban, hearts were perfused
for 60 min by recirculation with PSS containing 10 µCi/ml
32Pi after the stabilization period and
previously to the interventions assessed. At the end of the
experimental period, the ventricles were freeze-clamped, pulverized,
and stored at 70 °C until biochemical assay.
Preparation of SR Membrane Vesicles-- Membrane vesicles were prepared as described previously (13), except that the pulverized tissue from each heart was homogenized in 6 volumes of a medium containing 30 mM KH2PO4 (pH 7.0 at 4 °C), 5 mM Na2EDTA, 25 mM NaF, 300 mM sucrose, 1 mM phenylmethylsulfonyl fluoride, and 1 mM benzamidine. Samples from 32P-perfused hearts were homogenized in the same medium, except that the phosphate was replaced by 20 mM Tris-HCl (pH 7.0) at 4 °C. Protein was measured by the method of Bradford (19) using bovine serum albumin as a standard. The yield was 1-2 mg of membrane vesicle protein/g of cardiac tissue.
Electrophoresis and Western Blot Analysis-- SDS-polyacrylamide gel electrophoresis was performed using 10% acrylamide slab gels according to Porzio and Pearson (20) as described previously (13). Samples for electrophoresis were not boiled unless stated. For immunological detection of phospholamban phosphorylation sites, 10 µg of membrane protein were electrophoresed per gel lane. Proteins were transferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore Corp.) and probed as described previously (5) according to Drago and Colyer (21) with monoclonal antibody to phospholamban (1:5000) or polyclonal antibodies raised against a phospholamban peptide (residues 9-19) phosphorylated at Ser16 (1:5000) or at Thr17 (1:5000) (PhosphoProtein Research, Leeds, United Kingdom). Immunoreactivity was visualized by peroxidase-conjugated antibodies using a peroxidase-based chemiluminescence detection kit (Boehringer, Mannheim, Germany). The signal intensity of the bands on the film was quantified by optical densitometric analysis. To assess 32P incorporation into phospholamban, 300 µg of membrane protein were electrophoresed per gel lane. Gels were run in duplicate to use one of them for autoradiography and the other for liquid scintillation counting. Quantitative results are expressed as picomoles of 32P incorporated into phospholamban/mg of SR protein based on the specific activity of 32P in phosphocreatine (22).
Statistics-- All data are expressed as the mean ± S.E. of n preparations. Student's t test for unpaired observations was used to test for statistical differences. p < 0.05 was considered statistically significant.
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RESULTS |
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Effects of Acidosis on Phosphorylation of Phospholamban
Ser16 and Thr17 in the Absence and Presence of
-Adrenoreceptor Stimulation--
Fig.
1A shows the overall results
of 32P incorporation into phospholamban obtained from SR
membrane vesicles isolated from hearts perfused with 32P
and then in the absence and presence of 30 nM isoproterenol at pHo 7.40 and 6.80. In agreement with previous findings (16),
acidosis did not increase basal phospholamban phosphorylation, but
significantly enhanced isoproterenol-induced phospholamban phosphorylation. Fig. 1B shows immunoblots of SR membrane
vesicles obtained from hearts perfused under the same conditions of
Fig. 1A. Immunodetection of phosphorylation sites of
phospholamban indicated that the acidosis-induced increase in
phospholamban phosphorylation was exclusively due to an increase in
phosphorylation of Thr17. Similar results were obtained
when two other different isoproterenol concentrations were explored as
shown in Fig. 2. At all isoproterenol concentrations, acidosis significantly increased phosphorylation of
Thr17 of phospholamban without affecting phosphorylation of
Ser16. In the absence of isoproterenol, acidosis induced a
slight and nonsignificant increase in Thr17
phosphorylation and did not modify Ser16 phosphorylation.
Table I shows the mechanical parameters
of this experimental series. Acidosis induced a decrease in the maximal rate of contraction (+
) in both the absence and presence of isoproterenol. Moreover, acidosis produced an enhancement of the isoproterenol-induced relaxant effect (decrease in
t1/2), which attained significant levels at 30 nM isoproterenol. This relaxant effect of acidosis may be
due at least in part to the significant increase in the phosphorylation
of Thr17 produced by acidosis (see below). In the absence
of isoproterenol, the decrease in t1/2 produced by
acidosis was not associated with any significant increase in
phosphorylation of phospholamban and should therefore be attributed to
mechanisms unrelated to the phosphorylation of this protein, such as a
decrease in the calcium sensitivity of the myofilaments as suggested by
previous results (16).
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Does the Increase in [Ca]o Mimic the Acidosis Enhancement of Isoproterenol-induced Thr17 Phosphorylation?-- Fig. 3A shows immunoblots of SR membrane vesicles obtained from hearts perfused in the absence and presence of isoproterenol at [Ca]o = 1.35 mM and in the presence of isoproterenol at [Ca]o = 3.85 mM. Fig. 3B shows the overall results of 10 different experiments of this type. Increasing [Ca]o in the presence of isoproterenol significantly increased phosphorylation of Thr17. Unexpectedly, phosphorylation of Ser16 was significantly decreased. The opposite effects of increasing [Ca]o in the presence of isoproterenol on phosphorylation of phospholamban residues may explain the small effect of this maneuver on total phospholamban phosphorylation. As shown in Fig. 4, increasing [Ca]o from 1.35 to 3.85 mM evoked a small and nonsignificant increase in the phosphorylation of phospholamban induced by isoproterenol. Table I depicts the mechanical results of this experimental series. The increase in calcium supply to the cell in the presence of 30 nM isoproterenol did not modify the isoproterenol-induced increase in either contractility or relaxation. This result is consistent with the lack of effect of increasing [Ca]o in the presence of isoproterenol on total phospholamban phosphorylation as quantified by 32P incorporation into phospholamban (Fig. 4).
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Does the Inhibition of Phosphatases Mimic the Acidosis Enhancement of Isoproterenol-induced Thr17 Phosphorylation?-- Fig. 5A shows immunoblots of SR membrane vesicles obtained from hearts perfused at two different isoproterenol concentrations, 3 and 30 nM (pHo 7.40), in the absence and presence of 1 µM okadaic acid, a PP1 inhibitor. To compare the effects of okadaic acid with those of acidosis, SR membranes obtained from hearts perfused at 3 and 30 nM isoproterenol at pHo 6.80 were run in parallel. As already shown in Fig. 2, acidosis enhanced only isoproterenol-induced Thr17 phosphorylation without affecting Ser16 phosphorylation. In contrast, okadaic acid enhanced the isoproterenol-induced phosphorylation of both Ser16 and Thr17 at the two isoproterenol concentrations. Fig. 5B shows the mean values obtained by optic densitometric analysis of the different experiments of this series. These findings revealed that the sole inhibition of phosphatases by okadaic acid has a different effect on phosphorylation of Ser16 than that produced by acidosis. They also showed that even at the higher isoproterenol concentration used, which produced the maximum phosphorylation of phospholamban at pHo 7.40 (5), phosphatases are not maximally inhibited. Evidence for an enhancement of the isoproterenol-induced increase in phospholamban phosphorylation produced by okadaic acid has been previously reported in isolated myocytes (10). The present results further showed that this increase in phospholamban phosphorylation is due to the simultaneous increase in the phosphorylation of both Thr17 and Ser16 of phospholamban.
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Does Acidosis Increase Phospholamban Phosphorylation in the Absence
of Isoproterenol?--
The rationale behind this group of experiments
was to study the effects of stimulating the CaMKII pathway of
phospholamban phosphorylation in the absence of the inhibition of
phosphatases produced by cAMP-dependent mechanisms (7-11).
Previous experiments have shown that in the absence of isoproterenol,
the increase in [Ca]o did not phosphorylate phospholamban
unless the phosphatases were inhibited. This was so even when the
increase in contractility (and therefore cytosolic calcium) was similar to that evoked by isoproterenol or more prolonged as during tetani (5,
12-15). Thus, any increase in phospholamban phosphorylation produced
by increasing [Ca]o at low pHo would strongly suggest
a significant role of the acidosis-induced inhibition of phosphatases
in this effect. Fig. 6 shows the overall
results of the experiments performed in SR membrane vesicles from
hearts perfused with 32P and then at low and high
[Ca]o at pHo 7.40 and 6.80. The increase in
[Ca]o significantly increased phosphorylation of
phospholamban only under acidotic conditions. Fig.
7A shows immunoblots of SR
membrane vesicles isolated from hearts perfused as described for Fig.
6, except that 32P perfusion was omitted. Immunological
detection of the two phosphorylation sites of phospholamban showed that
the increase in phospholamban phosphorylation observed in Fig. 6 was
exclusively due to the phosphorylation of Thr17. The
overall results of this series are shown in Fig. 7B. Table I
shows the mechanical results of this experimental series. Acidosis produced a significant decrease in + and t1/2
at [Ca]o = 1.35 mM that was not associated with
any significant increase in phospholamban phosphorylation. At
[Ca]o = 3.85 mM, acidosis produced a decrease in
+
similar to that produced at low [Ca]o and a
significant decrease in t1/2 that occurred in
association with the increase in Thr17 phosphorylation.
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Contribution of Acidosis-induced Thr17 Phosphorylation
to the Relaxant Effect of Acidosis--
As shown in Table I, the
acidosis-induced relaxant effect at high [Ca]o in the absence
of isoproterenol was not greater than at [Ca]o = 1.35 mM, as would be expected from the significant increase in
Thr17 phosphorylation observed at high [Ca]o.
Similarly, the decrease in t1/2 induced by acidosis
in the presence of isoproterenol was not higher than that produced by
acidosis in the absence of the -agonist. To further explore this
point, additional experiments were performed in which the effect of
acidosis on Thr17 phosphorylation and
t1/2 was studied at high [Ca]o in the
presence and absence of a 10 µM concentration of the
CaMKII inhibitor KN-62. In these experiments, acidosis produced an
increase in Thr17 phosphorylation (expressed as percent of
the phosphorylation of Thr17 at 30 nM
isoproterenol, run in parallel) from 24.6 ± 8.3 to 82.4 ± 19.8%, which returned to control levels (28.6 ± 10.9%) in the presence of 10 µM KN-62 (n = 5). These
changes in Thr17 phosphorylation were paralleled by
changes in t1/2. The corresponding t1/2 values were 65.3 ± 4.1, 55.3 ± 4.4, and 66.0 ± 4.9 ms at high [Ca]o, high [Ca]o + acidosis, and high [Ca]o + acidosis + KN-62, respectively.
These results indicated that the increase in Thr17
phosphorylation evoked by acidosis was closely associated with a
relaxant effect. It is possible that at high
[Ca2+]i levels, as should occur at high
[Ca]o and in the presence of isoproterenol, the tension
developed by the contractile proteins may be close to or at the
"plateau" of the [Ca2+]i-tension curve. Under
these conditions, the acidosis-induced decrease in myofilament calcium
sensitivity and the resultant relaxant effect might be minimized. This
effect would be further minimized in the presence of isoproterenol by
the decrease in myofilament calcium sensitivity produced by the
-agonist.
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DISCUSSION |
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Phosphorylation site-specific antibodies have proven to be highly specific in the discrimination between the two sites of phosphorylation of phospholamban since no cross-reactivity with the other site of phosphorylation was observed (5). The combination of this technique with the quantification of 32P incorporation into phospholamban along with simultaneous measurements of mechanical parameters constitute invaluable tools to characterize the underlying mechanisms of phospholamban phosphorylation and their regulation.
Isoproterenol-induced phospholamban phosphorylation has been shown to
be dependent upon the acid-base status of the myocardium, with acidosis
enhancing the increase in phospholamban phosphorylation produced by the
-adrenergic agonist (Ref. 16 and our results in Fig. 1A).
The relevant findings of this study were that the increase in the
isoproterenol-induced phospholamban phosphorylation produced by
acidosis was exclusively due to an increase in phosphorylation of
Thr17 of phospholamban (Figs. 1B and 2) and that
this increase would contribute to the enhancement of the relaxant
effect of isoproterenol evoked by acidosis (Table I). The results also
provided evidence that an activation of CaMKII and an inhibition of
phosphatases may have both played a significant role in the observed
increase in Thr17 phosphorylation produced by acidosis in
the presence of isoproterenol.
Phosphorylation of phospholamban depends on a basic mechanism that is
common to any phosphorylation process, i.e. the relative activities of kinases and phosphatases that phosphorylate and dephosphorylate the protein, respectively. -Adrenoreceptor
stimulation increases phospholamban phosphorylation by increasing the
phosphorylation of both Ser16 and Thr17 (4, 5).
Different types of evidence further indicate that this dual
phosphorylation occurs because isoproterenol not only increases PKA
activity, but as a consequence of PKA activation, also produces a
simultaneous activation of CaMKII (by increasing [Ca2+]i) and inhibition of PP1, the major
phosphatase that dephosphorylates phospholamban (6-11). In this
context and in the search for the mechanisms underlying the enhancement
of the isoproterenol-induced phospholamban phosphorylation produced by acidosis, two possibilities should be necessarily explored: 1) acidosis
produces a further increase in CaMKII activity, and/or 2) acidosis
evokes a further inhibition of PP1. Both possibilities have some
experimental support (16-18). First, it has been shown in several
species that hypercapnic acidosis increases
[Ca2+]i more in the presence than in the absence
of isoproterenol (17, 18). This increase in
[Ca2+]i may add to the increase in
[Ca2+]i produced by isoproterenol to further
activate CaMKII. Second, it has also been shown that acidosis inhibits
PP1. A decrease in pHo from 7.40 to 6.80 produced by increasing
the CO2 of the gas mixture from 5 to 20% (external
bicarbonate of 18.5 mM) has been reported to produce a
decrease in pHi from 7.14 to ~6.70 in isolated cat and guinea
pig myocytes (23). Similar results were obtained by us in experiments
performed in rat isolated myocytes under the same conditions described
in the present experiments. In these experiments, the pHi
decreased from 7.18 ± 0.06 to 6.73 ± 0.04.2 In this pHi
range, we have previously shown in in vitro experiments that
PP1 was inhibited by acidosis by ~30% (16).
Previous results have shown that the increase in [Ca]o produced an increase in Thr17 phosphorylation only when the phosphatases were inhibited as in the presence of okadaic acid (5). In the present experiments, acidosis produced effects similar to those observed with okadaic acid: the activation of CaMKII by increasing [Ca]o in the absence of isoproterenol evoked an increase in Thr17 phosphorylation only under acidotic conditions (Fig. 7). This finding indicates that the acidosis-induced inhibition of phosphatases played a significant role in the increase in Thr17 phosphorylation observed. Moreover, the increase in Thr17 phosphorylation was closely paralleled by a relaxant effect. The fact that, at [Ca]o = 1.35 mM, acidosis did not produce any significant change in either Ser16 or Thr17 means that for the level of phosphatase inhibition produced by acidosis, the activity of kinases was too low to increase the hosphorylation of the corresponding residues.
The acidosis enhancement of isoproterenol-induced Thr17 phosphorylation could be mimicked by either increasing [Ca]o (Fig. 3) or adding okadaic acid (Fig. 5). These findings indicated that the CaMKII cascade can be activated above the level achieved by stimulation with isoproterenol and that even at the higher isoproterenol concentration used, phosphatases are not maximally inhibited by cAMP and therefore can be further inhibited by acidosis. However, neither the increase in [Ca]o nor the presence of okadaic acid mimicked the lack of effect of acidosis on Ser16. Whereas okadaic acid significantly enhanced the isoproterenol-induced phosphorylation of Ser16, the increase in [Ca]o significantly decreased it. The results obtained on phosphorylation of Ser16 under the different experimental situations are therefore consistent with the hypothesis that acidosis was acting by two different mechanisms, i.e. the increase in [Ca2+]i and the inhibition of phosphatases, both of which contribute to the increase in Thr17 phosphorylation, but have opposite effects on Ser16 phosphorylation.
The decrease in Ser16 phosphorylation when [Ca]o
was increased in the presence of isoproterenol (Fig. 3) was an
unexpected finding that deserves some additional comments. 1) The
result is in line with the fact that the increase in [Ca]o in the presence of the -agonist failed to significantly increase total
phospholamban phosphorylation (Fig. 4) and myocardial relaxation (Table
I) above the levels attained by
-adrenoreceptor stimulation. 2) As
discussed above, the result is also consistent with the idea that both
the activation of CaMKII and the inhibition of phosphatases have
contributed to the enhancement of the isoproterenol-induced Thr17 phosphorylation produced by acidosis. The cause for
this decrease in Ser16 phosphorylation is not apparent to
us. The inhibition of type V adenylyl cyclase, the major adenylyl
cyclase isoform present in adult ventricle (24), by submicromolar
concentrations of calcium has been previously described (25). However,
this possibility is not supported by previous experiments in our
laboratory showing that the increase in [Ca]o did not affect
intracellular cAMP levels (14). Another possible clue to explain the
above findings can be found in the mechanisms regulating PP1 activity. SR-associated PP1 could be inhibited by direct
PKA-dependent phosphorylation of the PP1 regulatory subunit
and PKA-dependent phosphorylation of inhibitor-1, which in
the phosphorylated state is a potent inhibitor of the catalytic subunit
of PP1 (8). In turn, the PP1 regulatory subunit and inhibitor-1 are
dephosphorylated by two other phosphatases, PP2A and PP2B (8). Since
PP2B is activated by calcium and calmodulin, the increase in
[Ca2+]i is a potential mechanism by which PP1 can
be activated (8). As a consequence, the inhibitory effect of PKA on PP1 would be attenuated. This phosphatase regulatory cascade might explain
the decrease in the phosphorylation of Ser16 when
[Ca2+]i was increased.
This decrease may not occur in acidosis if it is overrided by acidosis-induced phosphatase inhibition. The decrease in isoproterenol-induced Ser16 phosphorylation by increasing [Ca]o (Fig. 3) may therefore be the expression of a mechanism by which the increase in [Ca2+]i, acting through a protein phosphatase cascade, attenuates the signals that act via cAMP (8, 26).
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FOOTNOTES |
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* This work was supported by the Fundación Antorchas, the British Council, and the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina.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.
Established Investigator of the Consejo Nacional de
Investigaciones Científicas y Técnicas.
§ To whom correspondence should be addressed. Tel.: 54-21-83-4833; Fax: 54-21-25-5861; E-mail: cicme{at}isis.unlp.edu.ar.
1 The abbreviations used are: SR, sarcoplasmic reticulum; PKA, cAMP-dependent protein kinase; CaMKII, Ca2+/calmodulin-dependent protein kinase II; PSS, physiological salt solution; PP1, protein phosphatase type 1; PHL, phospholamban.
2 L. Vittone, C. Mundiña-Weilenmann, M. Said, and A. Mattiazzi, unpublished results.
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REFERENCES |
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