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Cellular Biology |
From the Laboratory of Cardiovascular Sciences (W.W., W.Z., S.W., D.Y., R.-P.X., H.C), National Institute on Aging, National Institutes of Health; Department of Medicine (M.T.C.), Johns Hopkins University, School of Medicine, Baltimore, Md; and The Institute of Molecular Medicine (R.-P.X., H.C.), Peking University, Beijing, China.
Correspondence to Heping Cheng, PhD, Laboratory of Cardiovascular Sciences, NIA, NIH, Baltimore, MD 21224. E-mail chengp{at}grc.nia.nih.gov
| Abstract |
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Key Words: ß1-adrenergic receptor Ca2+/calmodulin-dependent protein kinase II cAMP-dependent protein kinase cardiac contractility phospholamban
| Introduction |
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Recent studies have revealed unanticipated complexity of ßAR signal transduction. For ß2AR subtype stimulation in the heart, a parallel activation of Gi protein counterbalances Gs-mediated contractile response. Whereas ß1AR stimulated contractile response is thought to be mediated exclusively by the cAMP/PKA signaling pathway,8,9 ßAR regulation of cell growth and remodeling involves mitogen-activated protein kinase (MAPK) cascades and phosphoinositol 3 kinase (PI3K) pathway.10,11 Moreover, we have recently shown that sustained (24-hour) ß1AR stimulation progressively activates Ca2+/calmodulin-dependent protein kinase II (CaMKII), which is obligatory to cardiac apoptosis.12 Therefore, in addition to the classic cAMP/PKA pathway, chronic ßAR stimulation under certain physiological and pathophysiological circumstances may evoke pathways other than cAMP/PKA.13,14 These lines of evidence raise the question whether ß1AR modulates cardiac contractility using different sets of signaling mechanisms in short-term versus prolonged receptor stimulation.
CaMKII is a widely expressed protein kinase that modulates various functions ranging from learning and memory of the nervous system, muscle contraction, cell secretion to gene expression.15 In the heart, CaMKII
is the predominant isoform and plays a pivotal role in regulating cardiac performance and remodeling such as myocyte hypertrophy,16 apoptosis,12 and heart failure.17 Furthermore, CaMKII modulates an array of key proteins involved in cardiac excitation-contraction (E-C) coupling and Ca2+ handling, such as the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) and its regulator, phospholamban (PLB), ryanodine receptor (RyR) Ca2+ release channels, and sarcolemmal L-type Ca2+ channels (LCC).1822 However, the involvement of CaMKII in ß1AR modulation of myocardial contractility remains obscure.
The present study aimed at appraising roles of CaMKII and PKA in ß1AR modulation of cardiac E-C coupling, with an emphasis on the signaling mechanism for the sustained ß1AR stimulation. We found that both short-term and sustained ß1AR stimulation are efficacious in mediating positive inotropic and relaxant effects in cardiac myocytes. Unlike the short-term ß1AR stimulation, the sustained responses are mediated mainly by the CaMKII rather than the cAMP/PKA pathway. Furthermore, molecular integration of these two signaling pathways is mediated by dual site phosphorylation of key proteins involved in cardiac E-C coupling and its physiological regulation.
| Materials and Methods |
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1AR antagonist, prazosin (1 µmol/L), for 24 hours. All the antagonists were added at least 10 minutes before norepinephrine.
Dominant-Negative CaMKII
C Adenovirus Construction and Myocyte Infection
Dominant-negative CaMKII
C (DN-CaMKII) was generated by replacing the residue lysine43 with alanine (K43A) using the transformer site directed mutagenesis kit (Clontech). Adenoviral expression of ß-gal or the HA-tagged DN-CaMKII was performed at the multiplicity of infections (MOI) of 100. Twenty four hours after adenoviral infection, norepinephrine (100 nmol/L) was added.
Measurements of Cell Shortening and Ca2+ Transients
Measurements of cell contraction and Ca2+ transients were performed 24 hours after norepinephrine exposure. Normal cultured myocytes (24 hours) were used for short-term ß1AR stimulation. Myocytes were field-stimulated at 0.5 Hz in perfusion solution containing (in mmol/L) NaCl 137, KCl 4.9, CaCl2 1, MgSO4 1.2, NaH2PO4 1.2, glucose 15, and HEPES 20 (pH 7.4). Prazosin was added 10 minutes before short-term norepinephrine treatment. Protocols pertaining to specific experiments were given in the respective result figure
In indicator-unloaded myocytes, cell length was monitored by an optical edge tracking method at a 3-ms time resolution.23 In myocytes loaded with the Ca2+ indicator fluo-4/AM (Molecular Probes, 20 µmol/L for 30 minutes), Ca2+ transients and cell shortening were measured with a confocal laser scanning microscope (LSM510, Carl Zeiss). Digital image analysis used customer-designed programs coded in Interactive Data Language (IDL).
Receptor Radioligand Binding Assay
Cardiac myocytes were homogenized and crude membranes were prepared by centrifuging at 35 000g for 20 minutes at 4°C twice. ß1AR radioligand binding studies were performed in membranes (25 to 100 µg/tube) using the nonselective ßAR antagonist ligand 125I-cyanopindolol (125I-CYP, 1 to 300 pmol/L) as described previously.24 Nonspecific binding was determined in the presence of 10 µmol/L propranolol. The maximal numbers of binding sites (Bmax) and equilibrium dissociation constants (Kd) for 125I-CYP were determined by Scatchard analysis.
Immunostaining
Fixed cells were incubated with anti-HA antibody (1:500, Covance Research Products Inc) at 4°C overnight, followed by Cy5-conjugated secondary antibody (1:1000, Jackson ImmunoResearch laboratories). Negative controls were obtained by incubating cells with only the secondary antibody.
CaMKII Activity and cAMP Accumulation
Cell lysate (500 µg protein) was first immunoprecipitated with anti-CaMKII antibody (1:100, Santa Cruz Biotechnology) in a Ca2+-free medium. The precipitated proteins were evaluated with CaMKII assay kits (Upstate Biotechnology Inc) using a specific peptide substrate (KKALRRQETVDAL) as previously described.12 cAMP level was measured with the cAMP (3H) assay system (Amersham Biosciences) as described previously.24
Western Blotting Analysis of PLB Phosphorylation
PLB phosphorylation was detected with the phosphorylation site-specific antibodies recognizing P-16Ser PLB or P-17Thr PLB (1:10000, PhosphoProtein Research). Total PLB was detected with monoclonal antibody against PLB (Upstate Biotechnology).
Statistics
Data were reported as mean±SEM. Student t test and ANOVA with repeated measurement were applied, when appropriate, to determine statistical significance of the differences. P<0.05 was considered statistically significant.
| Results |
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1AR blocker, prazosin 1 µmol/L) for 24 hours. Cell shortening measured in the continued presence of ß1AR agonist was used as the end-point readout. Figure 1 shows typical examples and the average data of contractile response to short-term and sustained ß1AR stimulation. Sustained ß1AR stimulation (24 hours) elicited a 3.3-fold increase of the contraction amplitude (Figure 1C), which was accompanied by a 47% decrease of the 90% relaxation time (Figure 1D). Notably, the effects of sustained ß1AR stimulation on cell contraction and relaxation were fully reversible on washout of the agonist, as was the case with short-term ß1AR stimulation (Figure 1). That sustained contractile responses are fully and rapidly reversible excludes the possibility that they were due simply to a long-term "memory" formed during prolonged receptor stimulation.25
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As is the case with short-term ß1AR stimulation, sustained contractile response of ß1AR was sensitive to a nonselective ßAR blocker, propranolol (10 µmol/L), or a specific ß1AR blocker, CGP 20712A (0.5 µmol/L). Pretreatment with propranolol or CGP 20712A prevents the increase of contractility by subsequent 24-hour norepinephrine stimulation (Table 1); ICI 118,551 (0.5 µmol/L), a ß2AR specific antagonist, did not affect the 24-hour norepinephrine-induced response (data not shown). Radioligand binding assay of receptor density in crude membrane fraction revealed a trend of decrease (though statistically insignificant) in ß1AR density after 24-hour norepinephrine stimulation (Table 2), consistent with previous in vitro reports.26 When compared with the response to short-term ß1AR stimulation (10 minutes), we found that sustained ß1AR stimulation was equally efficacious in mediating the positive inotropic and relaxant effects (Figure 1). This observation is somewhat unexpected because it has been well established that cAMP/PKA signaling desensitizes within hours during prolonged ß1AR stimulation.5,27
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Sustained ß1AR Contractile Response Is Mediated by CaMKII Signaling Pathway
Whereas short-term and sustained ß1AR stimulations elicited indistinguishable responses in terms of cell contraction, there is no a priori reason that the same PKA-dependent mechanism is responsible for the contractile responses in both cases. To appraise role of PKA in mediating the sustained ß1AR responses, we used two inhibitors of the cAMP/PKA signaling: PKI, a membrane permeable peptide inhibitor of PKA, and Rp-cpt-cAMPS, an inhibitory cAMP analogue. We found that PKI treatment (10 µmol/L for 30 minutes) largely blocked the effect of short-term ß1AR stimulation (Figure 2A and 2B) as expected. By contrast, PKI was unable to reverse the increase in contraction amplitude in cells exposed to ß1AR stimulation for 24 hours (Figure 2A and 2B). Likewise, the cAMP antagonist Rp-cpt-cAMPS (100 µmol/L for 30 minutes) significantly inhibited the effects of short-term, but not sustained, ß1AR stimulation (Figure 2B). These results indicate that sustained contractile response of ß1AR stimulation is largely PKA-independent.
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Recently, it has been shown that sustained ß1AR-stimulated cardiomyocyte apoptosis requires activation of CaMKII signaling independently of PKA.12 Previous studies have also established an important role for CaMKII modulation of phosphorylation and function of key proteins involved in cardiac E-C coupling, including LCC,21,22 SERCA,18 and its regulator PLB,19 and RyR.20,28 Next, we examined the involvement of CaMKII signaling in the contractile response to sustained ß1AR stimulation. As shown in Figure 2C and 2D, the inotropic effect of sustained ß1AR stimulation was reversed by KN93 (10 µmol/L for 30 minutes), a synthetic CaMKII inhibitor, whereas the effect of short-term ß1AR stimulation was unaffected by KN93. KN92 (10 µmol/L), an inactive analogue of KN93, exerted no significant effects on either short-term or sustained ß1AR stimulation (data not shown). Similar to KN93, a cell-permeable peptide inhibitor of CaMKII, myristoylated autocamtide-2 related inhibitory peptide (AIP, 10 µmol/L for 30 minutes), completely abolished the positive inotropic effect of sustained ß1AR stimulation, without affecting those of short-term ß1AR stimulation (Figure 2D). Thus, inhibition of cAMP/PKA and CaMKII preferentially blocked contractile modulation by short-term and sustained ß1AR stimulation, respectively, indicating that the initial responses depend mainly on PKA, whereas the sustained responses require CaMKII signaling.
CaMKII-Dependent Increase of Ca2+ Transients in Response to Sustained ß1AR Stimulation
To further investigate cellular mechanisms underlying the contractile responses in sustained versus short-term ß1AR stimulation, we measured Ca2+ transients and the corresponding cell contraction in Ca2+ indicator-loaded myocytes, using confocal microscopy. Figure 3A shows typical micrographs of cellular Ca2+ transients and shortenings from cells that underwent short-term and sustained ß1AR stimulation in the absence or presence of PKA or CaMKII inhibitor. Both short-term and sustained ß1AR stimulation increased Ca2+ transient amplitude (
F/F0 from 2.7±0.1 to 5.5±0.2 or 6.0±0.2 for cells with short-term or sustained ß1AR stimulation, respectively, n=37 to 55 cells) and contractile amplitude (from 2.5±0.3% to 12.2±0.6% or 11.1±0.6% for cells with short-term or sustained ß1AR stimulation, respectively, n=33 to 55; Figure 3). The short-term ß1AR stimulation-induced Ca2+ and contractile responses were blocked by Rp-cpt-cAMPS but not by AIP (Figure 3). In contrast, the sustained ß1AR stimulationinduced increases in Ca2+ transients and contraction were resistant to Rp-cpt-cAMPS, but were reversed by AIP (Figure 3). These data indicate that both cAMP/PKA and CaMKII signaling pathways of ß1AR stimulation augment cell contraction by enhancing intracellular Ca2+ transients.
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Effects of Dominant-Negative CaMKII on Short-Term and Sustained ß1AR Stimulation
To confirm that the sustained ß1AR contractile effect is CaMKII-dependent, we resorted to molecular and genetic manipulation of the CaMKII signaling system. An adenovirus carrying the HA-tagged CaMKII gene with a dominant-negative mutation (DN-CaMKII, mutation K43A) that abrogates the kinase activity was constructed and infected cardiac myocytes in culture. Twenty-four hours after adenoviral infection at MOI of 100, nearly 100% cells showed intense immunostaining of DN-CaMKII as visualized by an anti-HA antibody (Figure 4A). Western Blotting analysis revealed that CaMKII-specific phosphorylation of PLB at 17Thr was also markedly reduced in the DN-CaMKII expressing myocytes (Figure 4B). Figure 4C and 4D show that sustained ß1AR stimulation in DN-CaMKII-expressing cells failed to elicit significant increases of Ca2+ transient amplitude or contraction amplitude. However, no significant difference between ß-gal and DN-CaMKII expression groups was found in contractile and Ca2+ responses to short-term ß1AR stimulation (Figure 4C and 4D). These data corroborate that sustained ß1AR stimulation enhances Ca2+ transients and cell contraction through a CaMKII pathway, whereas the initial ß1AR response is largely CaMKII-independent.
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Switch of cAMP and CaMKII Signaling During Sustained ß1AR Stimulation
To better characterize the aforementioned signaling shift during ß1AR stimulation, we directly measured cellular cAMP accumulation and CaMKII activation over a wide period of time (from 10 minutes up to 24 hours). ß1AR stimulation elicited a rapid increase of cellular cAMP production that reached its peak in 10 minutes. In the continued presence of the ß1AR agonist, however, cAMP production was reduced by 66% at 3 hours after stimulation (Figure 5A), and returned to basal level at the steady state (12 hours). The gradual decay of cAMP is consistent with the notion that cAMP/PKA signaling undergoes substantial desensitization during prolonged receptor stimulation.57
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Parallel measurement of CaMKII activity revealed a distinctly different temporal pattern for CaMKII response during ß1AR stimulation. CaMKII activity rose exponentially (time constant
=15 minutes) without an initial overshoot (Figure 5B). The plateau was reached after
1 hour stimulation and was stable for at least 24 hours. Overall, the gradual sensitization of CaMKII signaling roughly mirrored the desensitization of cAMP/PKA signaling, indicating a shift from a cAMP/PKA-dominant signaling to a CaMKII-dominant signaling. The slow and nondecremental activation of CaMKII provides the basis for the sustained contractile and Ca2+ responses to ß1AR stimulation.
Phospholamban (PLB) as a Molecular Integrator of ß1AR-Stimulated PKA and CaMKII Signals
The SR protein PLB, in its unphosphorylated form, serves as a constitutive inhibitor of the SR Ca2+ ATPase. PKA and CaMKII can independently phosphorylate PLB at 16Ser and 17Thr, respectively, and either site phosphorylation is sufficient to reverse its inhibition on SERCA activity and subsequently elicit positive inotropic and relaxant responses.29 Thus, PLB with its dual-site phosphorylation might operate as a molecular integrator of both short-term and sustained ß1AR signaling.
To explore this possibility, we examined the site-specific phosphorylation of PLB in response to sustained and short-term ß1AR stimulation. The phosphorylation at PKA-dependent site (P-16Ser) was increased by 7.2±0.9-fold (n=4, P<0.001 versus control) at 10 minutes after exposure to norepinephrine, but was then diminished toward the basal level during sustained ß1AR stimulation (Figure 5C). Conversely, phosphorylation at the CaMKII-dependent site (P-17Thr) was significantly increased in response to sustained, but not short-term, ß1AR stimulation (Figure 5D). These data support the idea that dual site phosphorylation by PKA and CaMKII in effector proteins (eg, PLB) serves to integrate the dual signaling pathways of ß1AR stimulation.
Effect of cAMP/PKA on CaMKII Mediated ß1AR Contractile Response
Because ß1AR-stimualted cAMP/PKA signaling precedes the CaMKII signaling, it might be argued that activation of CaMKII pathway in sustained ß1AR stimulation is still dependent on the initial PKA activation. However, preinhibition of cAMP/PKA with Rp-cpt-cAMPS (100 µmol/L) did not influence the sustained ß1AR-stimulated contractile and relaxant responses or the blockade effect of AIP (Figure 6A). Conversely, direct activation of cAMP/PKA pathway by forskolin (1 µmol/L), an adenylate cyclase activator, or cpt-cAMP (100 µmol/L), an active cAMP analogue, elicited no CaMKII-dependent component in the sustained inotropic response (Figure 6B and 6C). Thus, cAMP/PKA signaling appears to be neither sufficient nor necessary for ß1AR activation of CaMKII.
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| Discussion |
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Because inhibition of the cAMP/PKA pathway did not alter the responses to sustained receptor stimulation and receptor-independent cAMP/PKA signal failed to elicit CaMKII-dependent response (Figure 6), activation of the CaMKII pathway may not be consequential to the transient cAMP/PKA activation. In other words, different pathways initiated from the same GPCR may manifest desensitization or sensitization independently.6,12,30 As compared with ß1AR-stimulated CaMKII activation in ß1ß2AR double knockout mouse cardiac myocytes overexpressing ß1AR (
3-fold,
60 minutes), the elevation of CaMKII activity by native ß1AR in heart cells is modest (35% over baseline) yet exhibits faster kinetics (
15 minutes) (Figure 5B). These quantitative discrepancies might reflect differences in the receptor density or the coupling efficiency of ß1AR to downstream signaling pathways in these two systems.
Molecular Integration of ß1AR-Stimulated PKA and CaMKII Signals
That short-term and sustained ß1AR contractile and Ca2+ responses are virtually indistinguishable indicates a seamless integration of ß1AR-stimulated PKA and CaMKII signals at the molecular and cellular levels. Indeed, we detected that a shift of PLB phosphorylation from the PKA-dependent site (16Ser) in short-term ß1AR stimulation to the CaMKII-dependent site (17Thr), which correlates well with the time-dependent changes in cAMP production and CaMKII activation in sustained ß1AR stimulation. Because either site phosphorylation of PLB is sufficient to release its inhibition on the SERCA,28,31,32 PLB serves as a key molecular integrator of the dual signaling pathways of ß1AR stimulation. Disinhibition of SERCA activity by PLB phosphorylation will enhance SR Ca2+ recycling, accelerate relaxation of Ca2+ transients and cell contraction, and subsequently increase the SR Ca2+ load,12 contributing to both the positive inotropic and relaxant effects of ß1AR stimulation. It is noteworthy that the relaxant effect of sustained norepinephrine exposure was not significantly influenced by CaMKII inhibitors (Figure 2C), suggesting the possibility for a differential regulation of peak contraction and relaxation by sustained ß1AR stimulation.
In addition to PLB, previous studies have also shown that LCC Ca2+ currents are regulated by both PKA and CaMKII.21,33 Our preliminary data suggested the enhancement of LCC Ca2+ currents during initial (10 to 30 minutes) and sustained (3 to 6 hours) ß1AR stimulation is mediated by PKA- and CaMKII-dominant mechanisms, respectively, suggesting LCC serves as another molecular integrator of the PKA and CaMKII signals of ß1AR stimulation. In addition, it has been documented that either PKA or CaMKII can phosphorylate RyRs,20,34 although functional consequence of such phosphorylation remains controversial.3436
Functional Significance of ß1AR Signaling Switch
The finding of time-dependent switch between two signaling pathways of ß1AR stimulation bears important implications in understanding physiological modulation of cardiac function as well as the etiology and pathophysiology of cardiac diseases associated with chronically exaggerated ßAR signaling. First, the present result reassures that short-term ß1AR stimulation, as in fight-or-flight response or during exercise, is largely mediated by the cAMP/PKA pathway, rapid activation of which enables a beat-to-beat regulation of cardiac performance. However, the cAMP/PKA signaling desensitizes nearly completely (Figure 5A) and is unlikely to play any major role for more enduring responses. In contrast, the CaMKII signaling does not undergo any appreciable desensitization over the period of observation (up to 24 hours). If this can be extrapolated to chronic ß1AR stimulation in vivo, a corollary is that long-term ß1AR effects, beneficial or toxic, must be due primarily to CaMKII-dependent signal transduction. This concept resonates with several lines of evidence found in previous studies. First, ßAR-stimulated cardiac cell hypertrophy is largely PKA-independent but requires CaMKII activation.37,38 Second, CaMKII, but not PKA activation is obligatory to ß1AR-mediated cardiac apoptosis.12 Third, whole-animal and clinic data hint on that cardiac toxic effects of chronically enhanced ßAR stimulation are likely PKA-independent.39 Emerging evidence also suggests that increased CaMKII activity in human heart failure might play a compensatory role for the decreased cardiac contractility in failing hearts.40,41
Possible Mechanisms Underlying CaMKII Activation
The exact mechanisms underlying CaMKII activation and the switch of signaling pathways remain unknown. Preliminary observations showed that the ß1AR CaMKII-dependent response is insensitive to Gi/Go inhibition by pertussis toxin (1.5 µg/mL, added 3 hours before norepinephrine). Results in Figure 6 further suggest that direct and sustained activation of the cAMP/PKA signaling failed to activate CaMKII, and that blockage of cAMP/PKA does not prevent CaMKII from activation after ß1AR stimulation; thus, the ß1AR-cAMP signaling is neither necessary nor sufficient for the activation of ß1AR-induced CaMKII signaling. Apart from the cAMP/PKA pathway, some evidences support a direct coupling between LCC and GPCRs including ß2AR.42,43 Overexpression of Gs
has also been shown to activate LCC currents via PKA-independent mechanisms.44 If LCC could be activated in a PKA-independent manner, LCC Ca2+ entry might be responsible for the gradual CaMKII activation during ß1AR stimulation. In this scenario, the cAMP-dependent activation of LCC may differ from ß1AR-dependent, but cAMP-independent, LCC activation because sustained cAMP-dependent activation of LCC by forskolin or active cAMP analogue does not express the CaMKII signaling even after 24 hours. Recent studies have also hinted on a G proteinindependent GPCR signaling. For instance, the carboxyl terminus of ß1AR can directly interact with PDZ-motif containing proteins such as PSD-95 and Ras exchanger regulatory factor.45,46 By analogy, ß1AR might directly activate element(s) of the CaMKII pathway through G proteinindependent mechanisms. Future investigations are warranted to explore these possibilities.
In summary, ß1AR modulation of cardiac E-C coupling invokes dual signaling pathways mediated by cAMP/PKA and CaMKII, respectively. The cAMP/PKA signaling is biphasic with a prominent early peak, whereas the CaMKII signaling is slow but persistent. During the signaling switch, the inotropic and relaxant responses are maintained at the steady state because of the convergence of PKA and CaMKII signals onto common effector proteins involved in E-C coupling and intracellular Ca2+ regulation. As a result, the effects of sustained ß1AR stimulation (eg, inotropy, cell growth, and cell death) are due primarily to CaMKII, rather than PKA signaling. These findings provide mechanistic insights into ßAR modulation of cardiac function and GPCR signaling, and suggest new concepts for mechanistic understanding and therapeutic treatment of cardiac conditions such as hypertension and chronic heart failure that are associated with sustained elevation of endogenous catecholamines.
| Acknowledgments |
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| Footnotes |
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Original received April 26, 2004; revision received September 2, 2004; accepted September 7, 2004.
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