Published online before print May 30, 2002, doi: 10.1161/01.RES.0000024115.67561.54
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© 2002 American Heart Association, Inc.
Cellular Biology |
Phosphatidylinositol 3-Kinase Functionally Compartmentalizes the Concurrent Gs Signaling During ß2-Adrenergic Stimulation
From the Laboratory of Cardiovascular Science (S.-H.J., V.L., M.T.C., R.-P.X.), Gerontology Research Center, National Institute on Aging, National Institute of Health, Baltimore, Md; and the Department of Medicine and Biological Chemistry (P.H.W), Division of Endocrinology, Diabetes, and Metabolism, University of California, Irvine, Calif.
Correspondence to Rui-Ping Xiao, MD, PhD, Laboratory of Cardiovascular Science, Gerontology Research Center, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Dr, Baltimore, MD 21224. E-mail XiaoR{at}grc.nia.nih.gov
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Abstract |
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Compartmentation of intracellular signaling pathways serves as an important mechanism conferring the specificity of G protein-coupled receptor (GPCR) signaling. In the heart, stimulation of ß2-adrenoceptor (ß2-AR), a prototypical GPCR, activates a tightly localized protein kinase A (PKA) signaling, which regulates substrates at cell surface membranes, bypassing cytosolic target proteins (eg, phospholamban). Although a concurrent activation of ß2-AR-coupled Gi proteins has been implicated in the functional compartmentation of PKA signaling, the exact mechanism underlying the restriction of the ß2-AR-PKA pathway remains unclear. In the present study, we demonstrate that phosphatidylinositol 3-kinase (PI3K) plays an essential role in confining the ß2-AR-PKA signaling. Inhibition of PI3K with LY294002 or wortmannin enables ß2-AR-PKA signaling to reach intracellular substrates, as manifested by a robust increase in phosphorylation of phospholamban, and markedly enhances the receptor-mediated positive contractile and relaxant responses in cardiac myocytes. These potentiating effects of PI3K inhibitors are not accompanied by an increase in ß2-AR-induced cAMP formation. Blocking Gi or Gß
signaling with pertussis toxin or ßARK-ct, a peptide inhibitor of Gß, completely prevents the potentiating effects induced by PI3K inhibition, indicating that the pathway responsible for the functional compartmentation of ß2-AR-PKA signaling sequentially involves Gi, Gß, and PI3K. Thus, PI3K constitutes a key downstream event of ß2-AR-Gi signaling, which confines and negates the concurrent ß2-AR/Gs-mediated PKA signaling.
Key Words: ß2-adrenoceptor • cAMP signal compartmentation • phosphatidylinositol 3-kinase • phospholamban • cardiac contractility
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Introduction |
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Afundamental question of G protein-coupled receptor (GPCR) signal transduction is how a myriad of GPCRs and numerous cognate G proteins can elicit highly specific physiological responses. One of the most notable determinants of the specificity and effector-selectivity of receptor signaling is the cellular compartment over which the signal can transmit. As a prototypical GPCR, ß-adrenoceptor (AR) activates the classical Gs-adenylyl cyclase (AC)-cAMP-protein kinase A (PKA) signaling cascade. In the heart, PKA subsequently phosphorylates a multitude of regulatory proteins involved in cardiac muscle contraction, including sarcolemmal L-type Ca2+ channel,1 the sarcoplasmic reticulum (SR) Ca2+ pump regulator phospholamban (PLB),2 and myofilament proteins,3 resulting in increased contractility and accelerated cardiac relaxation.
However, a large body of evidence indicates that ß2-AR-induced cAMP/PKA signaling is tightly localized to the cell surface membrane microdomains in the vicinity of L-type Ca2+ channels and cannot transmit to non-sarcolemmal target proteins, whereas ß1-AR-mediated cAMP/PKA signaling can broadcast throughout the cell.4 Specifically, ß1-AR can activate sarcolemmal L-type Ca2+ channels and increase phosphorylation of multiple intracellular proteins, such as PLB at SR membrane and troponin I and C proteins of myofilaments, resulting in both positive contractile and relaxant responses.5–12 In contrast, ß2-AR stimulation selectively activates the Ca2+ channel, without affecting the aforementioned intracellular PKA target proteins, leading to a positive inotropic effect in the absence of a relaxant effect.4,7,13 Moreover, studies with patch-clamp single-channel recordings have shown that in both cardiac myocytes14 and hippocampal neurons,15 ß2-AR stimulation modulates single L-type Ca2+ channel activity only in a local mode (agonist included within the patch pipette with tip diameter 
1.0 µm) and not in a remote mode (agonist perfused outside the patch), whereas ß1-AR stimulation increases the channel activity in both modes.
The spatial and functional restriction of ß2-AR/Gs-induced cAMP/PKA signaling might be explained by the additional coupling of the receptor to Gi proteins.8,12,16 Inhibition of Gi signaling with pertussis toxin (PTX) allows ß2-AR stimulation to induce PLB phosphorylation5 and to modulate single L-type Ca2+ channels in the remote mode.14 Although the Gi pathway is implicated in the functional compartmentation of ß2-AR-mediated cAMP/PKA signaling, the downstream events of the ß2-AR-Gi pathway remain largely unknown.
Phosphoinositide 3-kinases (PI3Ks) are group of enzymes exhibiting both lipid kinase and protein kinase activities, and are broadly engaged in multiple vital cellular functions, including cell proliferation, cytoskeletal organization, cell migration, cell survival and cell growth.17–21 Based on their structure and substrate specificity, PI3Ks can be divided into 3 classes (I, II, and III), and the class I PI3Ks can be further categorized into 2 subclasses (A and B). The class IB PI3K (also known as PI3K) can be activated by Gß subunits of G proteins.22 Our recent studies have shown that PI3K constitutes a downstream mediator of ß2-AR-Gi signaling to deliver a cell survival signal, which protects heart muscle cells against a wide range of apoptotic insults such as hypoxia, reactive oxygen species, and enhanced ß1-AR stimulation.23,24 However, to date, there is no information available regarding the possible involvement of PI3K in acute modulation of cardiac function. Moreover, it remains unexplored whether PI3K participates in the Gi-dependent functional compartmentation of ß2-AR-PKA signaling, thereby reshaping the PKA-mediated protein phosphorylation and cardiac functional modulation.
The goal of the present investigation is to determine the possible involvement of PI3K in the functional restriction and effector-selectivity of the ß2-AR/Gs-mediated PKA signaling. We show that inhibition of PI3K by PI3K inhibitors enables ß2-AR-PKA signaling to reach intracellular proteins, as evidenced by a robust increase in PKA-dependent phosphorylation of PLB, a major regulator of cardiac relaxation, and that overexpression of constitutively active PI3K reduces basal phosphorylation of PLB. In the presence of the PI3K inhibitor, ß2-AR-induced PLB phosphorylation is associated with a de novo relaxant effect and an increase in the receptor-mediated positive contractile response. These potentiating effects induced by PI3K inhibition are fully prevented by disrupting Gi or Gß signaling, suggesting that the pathway linking Gi activation to the functional compartmentation of ß2-AR-activated PKA signaling sequentially involves Gi, Gß, and PI3K.
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Materials and Methods |
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Measurements of Cell Length and Intracellular Ca2+ Transients
Single ventricular myocytes were isolated from 2- to 4-month-old rat hearts by a standard enzymatic technique.25 Animals were provided by Charles River Laboratories (Wilmington, Mass), and the protocol of the study was approved by the Animal Care and Use Committee of the Gerontology Research Center, National Institute on Aging. In some experiments, heart cells were treated with 1.5 µg/mL PTX (Sigma) for 3 hours at 37°C to block Gi signaling, as described previously.8 In another subset experiments, myocytes were loaded with a fluorescent Ca2+ probe, indo-1/acetoxymethyl-ester (Molecular Probes), and then electrically paced at 0.5 Hz at 23°C, as described previously.25
PI3K Activity Assay
Suspensions of adult rat cardiomyocytes were first incubated with appropriate pharmacological agents. Measurement of PI3K activity was then performed, as previously described.23
Measurements of cAMP Accumulation and PKA-Dependent Phosphorylation of Phospholamban at Ser16
Intracellular cAMP was measured, as previously described.28 PKA-dependent phosphorylation of phospholamban (PLB) at Ser16 was assayed as described previously.5,10 Protein concentration was determined by the method of Lowry et al, 26 using ovalbumin as standard. After electrophoresis, the Western blot was performed as described previously.5,10
Culture and Adenoviral Infection of Adult Rat Ventricular Myocytes
In some experiments, heart cells were cultured and infected with adenovirus-p110* (constitutively active PI3K),27 adenovirus-ßARK-ct (an adenovirus vector encoding ßARK1 carboxyl-terminal domain, which blocks Gß signaling, kindly provided by R.J. Lefkowitz and W.J. Koch at Duke University, Durham, NC) or adenovirus-ß-gal (an adenovirus vector expressing lacZ, as a negative control), all at multiplicity of infection (moi) of 100, as previously described.24 All experiments were performed after 24 hours of adenoviral infection.
Statistics
All quantitative data are expressed as mean±SE. The results were analyzed using ANOVA. Differences were considered significant when values were P<0.05.
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ß2-AR Stimulation Increases PI3K Activity
To determine the role of PI3K in the functional compartmentation of ß2-AR signaling, we first examined the possible effect of ß2-AR stimulation on PI3K activity using a lipid kinase assay to monitor the conversion of PI into PI-P.23 As shown in Figure 1, ß2-AR stimulation by zinterol for 15 minutes increases PI-P production by
2.5-fold, and the addition of a highly selective ß1-AR antagonist, CGP20712A (0.3 µmol/L), does not alter this effect (data not shown). Stimulation of insulin-like growth factor-1 for 15 minutes, a known PI3K activator,29 is used as a positive control. LY294002 (5 µmol/L for 15 minutes), a PI3K inhibitor,30 completely blocks agonist-induced increase in PI3K activity. This result is consistent with our previous findings that ß2-AR stimulation activates PI3K in mouse and neonatal rat cardiac myocytes.23,24
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Inhibition of PI3K Enhances ß2-AR-Mediated Contractile Response
Next, we evaluated the potential involvement of PI3K in ß2-AR-mediated cardiac functional modulation with respect to single cell contraction and intracellular Ca2+ (Cai) handling. The PI3K inhibitor, LY294002, whereas it effectively blocks agonist-induced PI3K activation (Figure 1), has no significant effect on the amplitude and kinetics of baseline myocyte contraction (Table). However, LY294002 markedly enhances ß2-AR-mediated increase in cell contractility. Figure 2A shows a typical example of the ß2-AR agonist zinterol-elicited increase in contraction amplitude in the absence and presence of the PI3K inhibitor. The potentiating effect of LY294002 on ß2-AR contractile response occurs rapidly and is fully reversible on washout. Figure 2B illustrates the average dose-response of zinterol. The PI3K inhibitor shifts the whole dose-response curve upward and leftward, thus reducing the EC50 of zinterol from 3x10-7 to 5x10-8 mol/L.
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To further determine the possible effect of PI3K inhibition on ß2-AR-induced change in Cai transient, indexed by indo-1 fluorescent ratio,25 we simultaneously measured the Cai transient and contraction in freshly isolated adult rat cardiomyocytes loaded with indo-1 (Figure 2C). In the absence of LY294002, ß2-AR stimulation by zinterol (0.1 µmol/L, a concentration of EC50) increases the amplitudes of contraction and Cai transient to 162.3±13.2% and 124.4±4.0% of control, respectively (Figures 2C and 2D). Although LY294002 alone has no significant effect on either parameter (Table 1), it enhances zinterol-induced increases in the contraction and Cai transient by 2-fold and 1.5-fold, respectively (Figures 2C and 2D, also see Figures 7A and 2B). This result indicates that PI3K, known as a key molecule mediating the ß2-AR antiapoptotic effect,23,24 can acutely regulate cardiac contraction and Cai handling during ß2-AR stimulation.
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Inhibition of PI3K Enables ß2-AR Stimulation to Induce a De Novo Relaxant Effect
In addition to augmenting ß2-AR-induced positive inotropic effect, inhibition of PI3K by LY294002 overtly augments the effects of ß2-AR on the kinetics of contraction and Cai transient. As shown in Figure 2E, zinterol alone only slightly reduces the half relaxation time (t1/2) of contraction and has virtually no effect on the t1/2 of Cai transient, consistent with previous reports.5–8,13 However, in the presence of LY294002, ß2-AR stimulation causes a robust relaxant effect, as manifested by the significantly abbreviated t1/2 of both parameters (84.7±1.3% and 84.5±4.1% of control for contraction and Cai transient, respectively) (Figures 2C and 2E; also see Figures 7C and 7D). Because cardiac relaxant effect is a hallmark of PKA-dependent phosphorylation of PLB, the present data suggests that inhibition of PI3K permits ß2-AR stimulation to evoke a de novo relaxant effect, likely by affecting phosphorylation status of PLB, the major regulator of cardiac SR Ca2+ pump.2 To test this hypothesis, we next measured ß2-AR-induced PLB phosphorylation in the presence or absence of PI3K inhibition.
PI3K Inhibition Enables ß2-AR Stimulation to Increase Phosphorylation of PLB at Ser16
Previous studies have shown that PLB is phosphorylated by ß1-AR stimulation at 2 adjacent sites, Ser16 and Thr17, catalyzed by PKA and Ca2+/calmodulin-dependent kinase II, respectively,2,31 whereas ß2-AR stimulation is unable to induce phosphorylation of PLB and other cytosolic regulatory proteins.5,7,9,10 Consistent with the previous notion, the ß2-AR agonist, zinterol, has only a very minor effect on PKA-dependent phosphorylation of PLB at Ser16. A typical Western blot and the average dose-response relationship of ß2-AR-induced phosphorylation of PLB at Ser16 are shown in Figures 3A and 3B, respectively. Zinterol even at a maximal concentration (10 µmol/L) only increases Ser16-PLB phosphorylation by 1.2-fold. In sharp contrast, in LY294002-pretreated myocytes, ß2-AR stimulation by zinterol results in a dose-dependent increase in PLB phosphorylation, with a maximal effect of 3.8-fold increase and an EC50 of 3.2x10-7 mol/L. The inhibitory effect of PI3K on ß2-AR-induced PLB phosphorylation is further confirmed by using another PI3K inhibitor, wortmannin.32 Similar to LY294002, wortmannin also allows the ß2-AR agonist, zinterol, to increase PKA-dependent phosphorylation of PLB at Ser16 in a dose-dependent manner (Figures 3A and 3B). These results indicate that inhibition of PI3K enables ß2-AR to induce PLB phosphorylation at Ser16, thus resulting in a de novo relaxant effect. This provides the first evidence that PI3K is critically involved in the functional compartmentation and effector-selectivity of ß2-AR signaling.
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Overexpression of Constitutively Active PI3K Decreases Phosphorylation of PLB at Ser16
To further examine the possible role of PI3K on PKA-dependent PLB phosphorylation, we expressed a constitutively active PI3K (Adv-p110*) in cultured adult rat cardiomyocytes using adenoviral gene transfer. In myocytes overexpressing the constitutively active PI3K, basal phosphorylation of PLB at Ser16 is reduced by 50% as compared with that in myocytes-infected by a control virus (Adv-ß-gal, at the same moi) (Figures 4A and 4B), suggesting that activation of PI3K per se inhibits PKA-dependent phosphorylation of PLB. Interestingly, in myocytes expressing the constitutively active PI3K, pretreatment of cells with LY294002 (5 µmol/L for 10 minutes) cannot unmask ß2-AR-induced PLB phosphorylation, whereas it fully abolishes agonist-induced endogenous PI3K activation (Figure 1). This is consistent with the fact that LY294002 (5 µmol/L) is unable to inhibit the constitutively active PI3K-induced phosphorylation of Akt, a downstream effect of PI3K (data not shown, also see Wu et al27). This result indicates that the potentiating effect of LY294002 on ß2-AR-induced PLB phosphorylation at Ser16 is mediated specifically by inhibiting of PI3K activity.
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P<0.05 vs the baseline in cells infected with adenovirus-ß-gal).
Inhibition of PI3K by LY294002 Cannot Enhance ß2-AR-Induced cAMP Formation
To understand the mechanism underlying the potentiating effects of PI3K inhibition on ß2-AR-induced PLB phosphorylation, we next measured intracellular cAMP formation in response to ß2-AR stimulation in the presence and absence of the PI3K inhibitor, LY294002 (Figure 5). ß2-AR stimulation by zinterol (0.1 µmol/L, a concentration of EC50) significantly increases cAMP accumulation by 2-fold. However, pretreatment of cells with LY294002 cannot further enhance zinterol-induced increase in intracellular cAMP accumulation. This result suggests that the confining effect of ß2-AR-PI3K on the concurrent ß2-AR-Gs signaling is likely mediated by activating protein phosphatases, rather than by inhibiting formation of cAMP.
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Disruption of Gi Signaling and Inhibition of PI3K Have Nonadditive Effect on ß2-AR-Induced Phosphorylation of PLB at Ser16
We hypothesized that if PI3K activation is the downstream event of Gi signaling, disruption of Gi signaling by PTX treatment and inhibition of PI3K should have nonadditive effect on ß2-AR-induced phosphorylation of PLB at Ser16. Indeed, pretreatment of cells with both PTX and LY294002 exhibits no additive effect on PLB phosphorylation in response to the ß2-AR agonist, zinterol (0.1 µmol/L, a dose the EC50 used to avoid saturation). Cotreatment of cells with PTX and LY294002 enhances the response of PLB phosphorylation by 2.2-fold, similar to the single treatment of cells by either PTX or LY294002 (Figures 6A and 6B). The same conclusion is drawn when even a lower dose of zinterol (10 nmol/L) is utilized (1.8±0.4-, 1.9±0.4-, and 1.8±0.5-fold increase for PTX+zinterol, LY294002+zinterol, and PTX+LY294002+zinterol, respectively, n=6, P>0.05 among groups), indicating that the nonadditive effect is not due to a saturation of PLB phosphorylation. Taken together, these results strongly suggest that the additional Gi coupling offsets ß2-AR-stimulated PLB phosphorylation via a PI3K-dependent mechanism.
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Disruption of Gi Signaling Prevents the Potentiating Effects Induced by PI3K Inhibition
In PTX-treated cells, the PI3K inhibitor fails to further enhance the ß2-AR-induced augmentation in contraction amplitude (Figure 7A), consistent with the data on PLB phosphorylation described above. This is not caused by a saturation of cell contractile response, because the contraction amplitude can be increased by about 4-fold in response to a full ß-AR agonist, isoproterenol (1 µmol/L) (data not shown). Likewise, LY294002 does not significantly alter the response of Cai transient in those PTX-treated cells (Figure 7B). Furthermore, PTX treatment prevents LY294002 to potentiate the ß2-AR-mediated abbreviation in the t1/2 of contraction or Cai transient (Figures 7C and 7D). Thus, inhibition of Gi signaling completely abolishes the potentiating effects of the PI3K inhibitor on ß2-AR-mediated positive contractile and relaxant responses. This indicates that ß2-AR stimulation activates PI3K signaling via a Gi-dependent mechanism.
ß2-AR-Mediated PI3K Activation Requires Gß Signaling
We next sought to discriminate which subunit of heterotrimeric Gi proteins is essential to the PI3K activation by infecting myocytes with either adenovirus-ßARK-ct (ßARK-ct, a peptide inhibitor of Gß signaling)33 or adenovirus-ß-gal (as a negative control). It is noteworthy that the basal contraction or Cai transient in cells infected with adenovirus-ßARK-ct is comparable to that in myocytes infected by the control virus, adenovirus-ß-gal (Table). Figure 7E shows that in adenovirus-ß-gal-infected myocytes inhibition of PI3K by LY294002 augments zinterol-induced increase in the contraction amplitude, similar to the situation in freshly isolated myocytes (Figures 2 and 7A). However, in myocytes infected by adenovirus-ßARK-ct, LY294002 fails to further enhance ß2-AR-evoked positive inotropic effect. Similarly, inhibition of Gß signaling prevents LY294002 from potentiating the ß2-AR-mediated increase in the Cai transient (Figure 7F). These observations suggest that Gß subunits dissociated from Gi proteins are critically involved in ß2-AR-mediated activation of PI3K in cardiac myocytes.
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PI3K Functionally Restricts ß2-AR-Mediated cAMP/PKA Signaling
Our present study shows that inhibition of PI3K also enables ß2-AR to induce PLB phosphorylation as well as a de novo relaxant response in adult rat cardiomyocytes, and that overexpression of a constitutively active PI3K significantly decreases the basal phosphorylation of PLB. The potentiating effects caused by PI3K inhibition are completely prevented by blocking either Gi or Gß
signaling, indicating that the Gi-dependent restriction of ß2-AR-PKA signaling involves a Gi-Gß-PI3K pathway. Thus, in addition to its essential roles in the regulation of chronic cellular processes such as proliferation, cell migration, cell survival, and cell growth, PI3K constitutes an important molecular link responsible for the functional compartmentation of ß2-AR/Gs-PKA signaling, negating the ß2-AR/Gs-mediated phosphorylation of PKA target proteins and the positive cardiac contractile and relaxant responses.
Possible Mechanisms Underlying the Inhibitory Effect of PI3K on ß2-AR-PKA Signaling
The present result demonstrates that the PI3K inhibitor LY294002 markedly enhances the response of contraction or Cai transient to ß2-AR stimulation (Figure 2). This enhancement can be largely explained by the increase in PLB phosphorylation at Ser16 (Figure 3). It is widely accepted that increased PKA-dependent phosphorylation of PLB not only accelerates SR Ca2+ uptake, but also elevates SR Ca2+ content due to the increased Ca2+ pump activity,2 thereby leading to hastened cardiac relaxation and increased cardiac contractility. This is supported by the notion that in a PLB-deficient mouse model (a functional equivalent of PLB full phosphorylation), both cardiac contractility and relaxation velocity are significantly enhanced as compared with that in wild-type animals.34 Alternatively, inhibition of PI3K might enhance ß2-AR-induced increase in L-type Ca2+ currents, which could elevate the amplitudes of Ca2+ transient and contraction. However, it has been recently shown that activation of PI3K increases the L-type Ca2+ current in vascular smooth muscle cells.35 Nevertheless, the possible effect of PI3K on cardiac Ca2+ currents awaits future investigation.
It has been demonstrated that PI3K exerts both lipid kinase and protein kinase activities.17,18 Interestingly, the present results show that inhibition of PI3K markedly enhances ß2-AR-induced phosphorylation of PLB at Ser16 by PKA (Figure 3), suggesting that PI3K may cause dephosphorylation of phosphorylated PLB-Ser16. Because inhibition of PI3K by LY294002 does not affect zinterol-induced increase in cAMP formation (Figure 5) and inhibition of Gi signaling does not attenuate ß2-AR/Gs-induced increase in total cellular cAMP production13 or in PKA activity,5 the inhibitory effect of PI3K on ß2-AR-evoked PLB phosphorylation might occur downstream of cAMP/PKA, perhaps via direct or indirect activation of certain protein phosphatases. In this regard, emerging evidence suggests that ß2-AR is able to form a macromolecular signaling complex with protein phosphatase 2A.15 Moreover, calyculin A, an inhibitor of protein phosphatase 1 and 2A, potentiates ß2-AR-induced positive inotropic effect in a PTX-sensitive manner,5 suggesting that protein phosphatases might be engaged in ß2-AR/Gi signaling. In this scenario, PI3K might act as a protein kinase to increase protein phosphatase activity directly via phosphorylation or indirectly through phosphorylation and subsequent inactivation of endogenous protein phosphatase inhibitor-I. Further study is merited to address these issues.
Regarding the involvements of specific PI3K isoenzymes in ß2-AR/Gi signaling, previous studies have shown that the class IB PI3K (PI3K isoform) is a downstream target of Gß signaling.22 However, recent studies suggest that in cultured neonatal rat cardiac myocytes, stimulation of ß-AR (subtype not specified) may activate PI3K
and ß.36 Because of the lack of isoform-specific PI3K inhibitors, the present study cannot discriminate the specific isoenzymes of PI3K family involved in ß2-AR-coupled Gi signaling in adult rat cardiac myocytes.
Potential Involvement of PI3K in Agonist-Induced ß2-AR Desensitization and Its Pathophysiological Relevance
As is true for all of GPCRs, the exposure of a receptor to an agonist results in rapid diminishment in its signaling efficiency (referred to as desensitization). Agonist-dependent ß2-AR desensitization is initiated by phosphorylation of the activated receptor by members of the GPCR kinase family, particularly the ß-adrenergic receptor kinase-1 (ßARK1).37 It is noteworthy that PI3K is able to physically associate with ßARK1 and promote agonist-dependent ß-AR internalization,38 perhaps contributing to agonist-dependent ß-AR desensitization. The robust inhibitory effect of PI3K activation on ß2-AR-induced positive contractile and relaxant responses, demonstrated by the present results, raises an intriguing and important question regarding the possible requirement of PI3K for agonist-induced ß2-AR desensitization.
Although activation of ß2-AR/Gi-mediated PI3K pathway protects cardiac myocytes against apoptosis,23,24 an imbalance of ß2-AR-initiated Gs and Gi signaling cascades may have pathological consequences. Both recent in vivo and in vitro studies have demonstrated that activation of PI3K signaling is coincident with heart muscle cell hypertrophy in response to pressure overload or ß-AR stimulation.36,39 In addition, in many kinds of chronic heart failure in human and animal models, the contractile response to ß-AR stimulation is markedly diminished,40,41 which is accompanied by enhanced Gi signaling.42 It is speculated that the upregulation of ß2-AR-Gi signaling in the functionally compensated hypertrophic heart or in the early stages of heart failure may protect against myocyte apoptosis and consequently slow the progression of cardiomyopathy and contractile dysfunction. However, the exaggerated ß2AR-Gi signaling may blunt the Gs-mediated contractile support, contributing to the phenotype of decompensated heart failure.
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Acknowledgments |
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This work is supported by NIH intramural research grant (R.-P.X.). The authors would like to thank Drs H. Cheng and E.G. Lakatta for stimulating discussions, and Dr H. Spurgeon and B. Ziman for their excellent technique support.
Received February 1, 2002; revision received May 20, 2002; accepted May 20, 2002.
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