Gi Protein-mediated Functional Compartmentalization of Cardiac β2-Adrenergic Signaling
1999; Elsevier BV; Volume: 274; Issue: 31 Linguagem: Inglês
10.1074/jbc.274.31.22048
ISSN1083-351X
AutoresMeike Kuschel, Ying-Ying Zhou, Heping Cheng, Sheng-Jun Zhang, Ye Chen‐Izu, Edward G. Lakatta, Rui-Ping Xiao,
Tópico(s)Receptor Mechanisms and Signaling
ResumoIn contrast to β1-adrenoreceptor (β1-AR) signaling, β2-AR stimulation in cardiomyocytes augmentsl-type Ca2+ current in a cAMP-dependent protein kinase (PKA)-dependent manner but fails to phosphorylate phospholamban, indicating that the β2-AR-induced cAMP/PKA signaling is highly localized. Here we show that inhibition of Gi proteins with pertussis toxin (PTX) permits a full phospholamban phosphorylation and a de novo relaxant effect following β2-AR stimulation, converting the localized β2-AR signaling to a global signaling mode similar to that of β1-AR. Thus, β2-AR-mediated Gi activation constricts the cAMP signaling to the sarcolemma. PTX treatment did not significantly affect the β2-AR-stimulated PKA activation. Similar to Gi inhibition, a protein phosphatase inhibitor, calyculin A (3 × 10−8m), selectively enhanced the β2-AR but not β1-AR-mediated contractile response. Furthermore, PTX and calyculin A treatment had a non-additive potentiating effect on the β2-AR-mediated positive inotropic response. These results suggest that the interaction of the β2-AR-coupled Gi and Gs signaling affects the local balance of protein kinase and phosphatase activities. Thus, the additional coupling of β2-AR to Giproteins is a key factor causing the compartmentalization of β2-AR-induced cAMP signaling. In contrast to β1-adrenoreceptor (β1-AR) signaling, β2-AR stimulation in cardiomyocytes augmentsl-type Ca2+ current in a cAMP-dependent protein kinase (PKA)-dependent manner but fails to phosphorylate phospholamban, indicating that the β2-AR-induced cAMP/PKA signaling is highly localized. Here we show that inhibition of Gi proteins with pertussis toxin (PTX) permits a full phospholamban phosphorylation and a de novo relaxant effect following β2-AR stimulation, converting the localized β2-AR signaling to a global signaling mode similar to that of β1-AR. Thus, β2-AR-mediated Gi activation constricts the cAMP signaling to the sarcolemma. PTX treatment did not significantly affect the β2-AR-stimulated PKA activation. Similar to Gi inhibition, a protein phosphatase inhibitor, calyculin A (3 × 10−8m), selectively enhanced the β2-AR but not β1-AR-mediated contractile response. Furthermore, PTX and calyculin A treatment had a non-additive potentiating effect on the β2-AR-mediated positive inotropic response. These results suggest that the interaction of the β2-AR-coupled Gi and Gs signaling affects the local balance of protein kinase and phosphatase activities. Thus, the additional coupling of β2-AR to Giproteins is a key factor causing the compartmentalization of β2-AR-induced cAMP signaling. The classical view of β-AR 1The abbreviations used are: β1-AR, β1-adrenoreceptor; PLB, phospholamban; PTX, pertussis toxin; PKA, cAMP-dependent protein kinase; NE, norepinephrine. signal transduction is that agonist-bound β-AR selectively interact with stimulatory G proteins (Gs), which, in turn, activate adenylyl cyclase to enhance cAMP formation. Subsequently, PKA phosphorylates a multitude of regulatory proteins involved in cardiac excitation-contraction coupling, including l-type Ca2+ channels (1Bean B.P. Nowycky M.C. Tsien R.W. Nature. 1984; 307: 371-375Crossref PubMed Scopus (250) Google Scholar,2Kameyama M. Hofmann F. Trautwein W. Pfluegers Arch. 1985; 405: 285-293Crossref PubMed Scopus (265) Google Scholar), the sarcoplasmic reticulum Ca2+ pump regulator PLB (3Wegener A.D. Simmerman H.K. Lindemann J.P. Jones L.R. J. Biol. Chem. 1989; 264: 11468-11474Abstract Full Text PDF PubMed Google Scholar,4Mundina-Weilenmann C. Vittone L. Ortale M. de Cingolani G.C. Mattiazzi A. J. Biol. Chem. 1996; 271: 33561-33567Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), and myofilament proteins (5England P.J. Biochem. J. 1976; 160: 295-304Crossref PubMed Scopus (116) Google Scholar, 6Garvey J.L. Kranias E.G. Solaro R.J. Biochem. J. 1988; 249: 709-714Crossref PubMed Scopus (168) Google Scholar), producing positive inotropic and lusitropic effects. In addition, PKA also phosphorylates and activates the endogenous protein phosphatase inhibitor 1 (7Neumann J. Gupta R.C. Schmitz W. Scholz H. Nairn A.C. Watanabe A.M. Circ. Res. 1991; 69: 1450-1457Crossref PubMed Scopus (86) Google Scholar), which further ensures the action of protein kinases by protein phosphatase inhibition. Although cardiac β1-AR signaling follows the scheme described above, recent studies have revealed a dissociation of β2-AR-mediated positive inotropic as well as lusitropic effects from global cAMP accumulation in several mammalian species (8Xiao R.P. Hohl C. Altschuld R. Jones L. Livingston B. Ziman B. Tantini B. Lakatta E.G. J. Biol. Chem. 1994; 269: 19151-19156Abstract Full Text PDF PubMed Google Scholar, 9Altschuld R.A. Starling R.C. Hamlin R.L. Billman G.E. Hensley J. Castillo L. Fertel R.H. Hohl C.M. Robitaille P.M. Jones L.R. Xiao R.P. Lakatta E.G. Circulation. 1995; 92: 1612-1618Crossref PubMed Scopus (126) Google Scholar, 10Zhou Y.Y. Cheng H. Bogdanov K.Y. Hohl C. Altschuld R. Lakatta E.G. Xiao R.P. Am. J. Physiol. 1997; 273: H1611-H1618PubMed Google Scholar, 11Kuschel M. Zhou Y.Y. Spurgeon H.A. Bartel S. Karczewski P. Zhang S.J. Krause E.G. Lakatta E.G. Xiao R.P. Circulation. 1999; 99: 2458-2465Crossref PubMed Scopus (106) Google Scholar). In addition, it has been demonstrated that in contrast to β1-AR, β2-AR stimulation fails to induce a cAMP-dependent phosphorylation of non-sarcolemmal proteins involved in excitation-contraction coupling and energy metabolism (e.g. phospholamban, the myofilament proteins, troponin I, C protein, and the cytosolic protein glycogen phosphorylase kinase), but it does activate sarcolemmal l-type Ca2+channels (10Zhou Y.Y. Cheng H. Bogdanov K.Y. Hohl C. Altschuld R. Lakatta E.G. Xiao R.P. Am. J. Physiol. 1997; 273: H1611-H1618PubMed Google Scholar, 11Kuschel M. Zhou Y.Y. Spurgeon H.A. Bartel S. Karczewski P. Zhang S.J. Krause E.G. Lakatta E.G. Xiao R.P. Circulation. 1999; 99: 2458-2465Crossref PubMed Scopus (106) Google Scholar, 12Xiao R.P. Lakatta E.G. Circ. Res. 1993; 73: 286-300Crossref PubMed Scopus (210) Google Scholar), suggesting that β2-AR signaling is localized. More direct evidence supporting the localized β2-AR signaling has emerged from singlel-type Ca2+ channel recordings. Employing cell-attached patch clamp technique, the activity of singlel-type Ca2+ channels has been measured in response to specific β-AR subtype agonist outside (remote) or inside (local) the patch pipette. In contrast to the diffusive effect of β1-AR stimulation, β2-AR stimulation by zinterol only locally activates the l-type Ca2+channel but has no remote effect (13Chen-Izu Y. Xiao R.P. Spurgeon H. Lakatta E.G. J. Mol. Cell. Cardiol. 1998; 30 (abstr.): 502Google Scholar). Taken together, these previous studies have led to the hypothesis that the β2-AR-induced cAMP signaling is compartmentalized to a subsarcolemmal space and cannot be transmitted to cytoplasmic and SR PKA target proteins. Alternatively, the signal is transmitted to cytosolic proteins but local inactivation occurs at these sites. The mechanism for the local control of β2-AR mediated signaling remains unclear. In many biological systems, Gsand Gi proteins cross-talk and operate as a complementary system. This balance system is usually regulated through different receptor families. For instance, activation of muscarinic receptors or adenosine receptors, prototypic Gi-coupled receptors, markedly antagonizes the positive inotropic effect of β-AR stimulation (14Levy M.N. Martin P.J. Stuesse S.L. Annu. Rev. Physiol. 1981; 43: 443-453Crossref PubMed Scopus (63) Google Scholar, 15Endoh M. Yamashita S. Eur. J. Pharmacol. 1980; 65: 445-448Crossref PubMed Scopus (63) Google Scholar). Interestingly, promiscuous G protein coupling of β2-AR to both Gs and PTX-sensitive G proteins (Gi2 and Gi3) has been demonstrated in intact cardiomyocytes (16Xiao R.P. Avdonin P. Zhou Y.Y. Cheng H. Akhter S.A. Eschenhagen T. Lefkowitz R.J. Koch W.J. Lakatta E.G. Circ. Res. 1999; 84: 43-52Crossref PubMed Scopus (337) Google Scholar, 17Xiao R.P. Ji X. Lakatta E.G. Mol. Pharmacol. 1995; 47: 322-329Crossref PubMed Scopus (201) Google Scholar). This coupling of β2-AR to Gi proteins and its downstream pathway might interplay with the β2-AR/Gs signaling and contribute to the localization of β2-AR signaling near the sarcolemmal membrane. Thus, in the present study, we intended to determine whether the localized β2-AR signaling is mediated by the additional coupling of β2-AR to Gi proteins and, if so, to elucidate potential underlying mechanisms. Single ventricular myocytes were isolated from rat hearts by a standard enzymatic technique (18Spurgeon H.A. Stern M.D. Baartz G. Raffaeli S. Hansford R.G. Talo A. Lakatta E.G. Capogrossi M.C. Am. J. Physiol. 1990; 258: H574-H586PubMed Google Scholar). The cells were suspended in HEPES pH 7.4 buffer containing (in mmol/liter) 20 HEPES, 1 CaCl2, 137 NaCl, 5 KCl, 15 dextrose, 1.3 MgSO4, 1.2 NaH2PO4. In some experiments, cells were separately or simultaneously treated with 1.5 μg/ml PTX (Sigma) for 3 h at 37 °C to block Gi protein activation (15Endoh M. Yamashita S. Eur. J. Pharmacol. 1980; 65: 445-448Crossref PubMed Scopus (63) Google Scholar) or 3 × 10−8m calyculin A (Calbiochem) for 20 min at 23 °C to inhibit protein phosphatases. Cells were stimulated at 0.5 Hz at 23 °C, and cell length was monitored from the bright-field image by an optical edge-tracking method using a photodiode array (model 1024 SAQ, Reticon) with a 3-ms time resolution (18Spurgeon H.A. Stern M.D. Baartz G. Raffaeli S. Hansford R.G. Talo A. Lakatta E.G. Capogrossi M.C. Am. J. Physiol. 1990; 258: H574-H586PubMed Google Scholar). Whole cell L-type Ca2+ current (ICa) was measured via patch clamp technique using an Axopatch 1D amplifier (Axon Instruments Inc.) (12Xiao R.P. Lakatta E.G. Circ. Res. 1993; 73: 286-300Crossref PubMed Scopus (210) Google Scholar). To activate ICa selectively, cells were voltage-clamped at −40 mV to inactivate Na+ and T-type Ca2+ channels. K+ currents were inhibited by 4 mmol/liter 4-aminopyridine and 5.4 mmol/liter CsCl instead of KCl in the HEPES buffer and the pipette solution containing (mmol/liter) CsCl 100, NaCl 10, tetraethanolamine Cl 20, HEPES 10, MgATP 5, EGTA 5, pH 7.2, adjusted with CsOH. ICa was elicited by 300-ms pulses from a holding potential of −40 to 0 mV at 0.5 Hz at 23 °C. ICa was measured as the difference between the current at the peak and the end of the 300-ms pulse. The detection of site-specific PLB phosphorylation was performed as described recently (11Kuschel M. Zhou Y.Y. Spurgeon H.A. Bartel S. Karczewski P. Zhang S.J. Krause E.G. Lakatta E.G. Xiao R.P. Circulation. 1999; 99: 2458-2465Crossref PubMed Scopus (106) Google Scholar). Briefly, cardiomyocytes were treated for 10 min with specific β-AR subtype agonists as indicated and solubilized prior to electrophoresis at 95 °C for 5 min to dissociate fully PLB into its monomeric form. Following electrophoresis, proteins were transferred to a polyvinylidene difluoride membrane (Serva), which was probed with the phosphorylation site-specific Ser16 PLB antibody (PhosphoProtein Research). Following incubation with a peroxidase-conjugated antibody (Dianova), the immunoreaction was detected with ECL (Amersham Pharmacia Biotech) and quantified with a video documentation system (Bio-Rad). The activation of PKA in soluble and particulate fractions was analyzed by a modified method of Murrayet al. (19Murray K.J. England P.J. Lynham J.A. Mills D. Schmitz-Pfeiffer C. Reeves M. Biochem. J. 1990; 267: 703-708Crossref PubMed Scopus (30) Google Scholar). Rat cardiomyocytes were homogenized and centrifuged at 6,000 × g for 5 min. The resulting supernatant was taken to represent the soluble protein kinase activity and the resuspended pellet, the particulate fraction. The PKA activity is expressed as the activity ratio of malantide (Bachem)32P incorporation in the absence and presence of cAMP (2.8 μmol/liter). Results are presented as means ± S.E. Statistical significance was determined by Student's t test or analysis of variance when appropriate. Values with p< 0.05 were considered to be statistically significant. The β2-AR agonist, zinterol (10−5m), increased the whole cell l-type Ca2+ current (ICa) to 161 ± 8.8% (n = 7, p < 0.05) of control in single rat cardiomyocytes (Fig. 1a), which was completely abolished by the β2-AR antagonist ICI 118,551 (10Zhou Y.Y. Cheng H. Bogdanov K.Y. Hohl C. Altschuld R. Lakatta E.G. Xiao R.P. Am. J. Physiol. 1997; 273: H1611-H1618PubMed Google Scholar). To delineate a role of cAMP-dependent PKA activation in the modulation of l-type Ca2+channels, an inhibitory cAMP analog, (Rp)-cAMPs (10Zhou Y.Y. Cheng H. Bogdanov K.Y. Hohl C. Altschuld R. Lakatta E.G. Xiao R.P. Am. J. Physiol. 1997; 273: H1611-H1618PubMed Google Scholar, 11Kuschel M. Zhou Y.Y. Spurgeon H.A. Bartel S. Karczewski P. Zhang S.J. Krause E.G. Lakatta E.G. Xiao R.P. Circulation. 1999; 99: 2458-2465Crossref PubMed Scopus (106) Google Scholar), was used to specifically block PKA activation. In the presence of (Rp)-cAMPs (10−4m) zinterol failed to augment ICa (Fig. 1b), indicating that the cAMP-dependent PKA activation is obligatory for β2-AR-mediated modulation ofl-type Ca2+ channels. PLB, the main modulator of cardiac relaxation, is phosphorylated following cardiac β1-AR stimulation at two adjacent phosphorylation sites, Ser16 (Fig. 2a) and Thr17, catalyzed by PKA and Ca2+/calmodulin-dependent kinase, respectively (3Wegener A.D. Simmerman H.K. Lindemann J.P. Jones L.R. J. Biol. Chem. 1989; 264: 11468-11474Abstract Full Text PDF PubMed Google Scholar, 4Mundina-Weilenmann C. Vittone L. Ortale M. de Cingolani G.C. Mattiazzi A. J. Biol. Chem. 1996; 271: 33561-33567Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 20Koss K.L. Kranias E.G. Circ. Res. 1996; 79: 1059-1063Crossref PubMed Scopus (300) Google Scholar). In contrast, the β2-AR agonist, zinterol, even at a maximal concentration (10−5m for 10 min), had only a very minor effect on the PKA-mediated Ser16 phosphorylation of PLB, as detected with phosphorylation site-specific PLB antibodies (21Drago G.A. Colyer J. J. Biol. Chem. 1994; 269: 25073-25077Abstract Full Text PDF PubMed Google Scholar) in the Western blot (Fig. 2, a and b). The dose-response relation and the time course of Ser16 PLB phosphorylation are shown in Fig. 2b and Fig. 3c, respectively. A maximal concentration (10−5m) of the β2-AR agonist, zinterol, only induced a minor increase in Ser16 PLB phosphorylation even if the incubation time was extended to 20 min (Fig. 3c). Concomitantly, the β2-AR-mediated increase in contractility occurred in the absence of a significant relaxant effect (Fig. 3a). Thus, the failure of β2-AR stimulation to induce PLB phosphorylation is the apparent mechanism for the absence of a lusitropic effect (Fig. 3c). These data illustrate that whereas both β1- and β2-AR share the common second messenger cAMP, they exhibit differences with respect to PKA-mediated protein phosphorylation and relaxant effects (8Xiao R.P. Hohl C. Altschuld R. Jones L. Livingston B. Ziman B. Tantini B. Lakatta E.G. J. Biol. Chem. 1994; 269: 19151-19156Abstract Full Text PDF PubMed Google Scholar, 9Altschuld R.A. Starling R.C. Hamlin R.L. Billman G.E. Hensley J. Castillo L. Fertel R.H. Hohl C.M. Robitaille P.M. Jones L.R. Xiao R.P. Lakatta E.G. Circulation. 1995; 92: 1612-1618Crossref PubMed Scopus (126) Google Scholar, 10Zhou Y.Y. Cheng H. Bogdanov K.Y. Hohl C. Altschuld R. Lakatta E.G. Xiao R.P. Am. J. Physiol. 1997; 273: H1611-H1618PubMed Google Scholar, 11Kuschel M. Zhou Y.Y. Spurgeon H.A. Bartel S. Karczewski P. Zhang S.J. Krause E.G. Lakatta E.G. Xiao R.P. Circulation. 1999; 99: 2458-2465Crossref PubMed Scopus (106) Google Scholar, 12Xiao R.P. Lakatta E.G. Circ. Res. 1993; 73: 286-300Crossref PubMed Scopus (210) Google Scholar).Figure 3PTX treatment selectively enhances the β2-AR-mediated augmentation of contraction amplitude, accelerates relaxation, and rescues Ser16 PLB phosphorylation. a, the time course of β2-AR (zinterol (Zin), 10−5m, 10 min)-mediated positive inotropic and relaxant effects in the presence (+ PTX) and absence of PTX (− PTX) (n = 10 cells for each data point). b, β1-AR stimulation (NE, 10−7m and 10−6mprazosin)-induced positive inotropic and relaxant effects are not affected by PTX (n = 10 cells for each data point).c, the time course of the β2-AR (Zin, 10−5m)-mediated de novo Ser16 PLB phosphorylation (n = 4–15) correlates with the relaxation effect in PTX-treated cardiomyocytes. Base-line values for contraction amplitude are 5.9 ± 0.5 (n = 20) and 6.9 ± 0.5 (n= 20) and for t½ are 378.9 ± 10.7 ms (n = 20) and 356.9 ± 19.4 ms (n = 20) in the absence or presence of PTX, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Based on our recent finding that β2-AR dually couples to Gs and Gi proteins (16Xiao R.P. Avdonin P. Zhou Y.Y. Cheng H. Akhter S.A. Eschenhagen T. Lefkowitz R.J. Koch W.J. Lakatta E.G. Circ. Res. 1999; 84: 43-52Crossref PubMed Scopus (337) Google Scholar, 17Xiao R.P. Ji X. Lakatta E.G. Mol. Pharmacol. 1995; 47: 322-329Crossref PubMed Scopus (201) Google Scholar), we hypothesized that the additional coupling of β2-AR to Gimight interfere with the β2-AR/Gs signaling, contributing to the restriction of β2-AR-mediated cAMP signaling to a subsarcolemmal domain. To test this hypothesis, we examined the effect of Gi protein inhibition by PTX on the PLB phosphorylation following β2-AR stimulation and its functional consequences. Whereas PTX itself had only a negligible effect on the basal Ser16 PLB phosphorylation (Fig. 2b), β2-AR stimulation with zinterol in PTX-treated cardiomyocytes markedly increased PLB phosphorylation in a dose-dependent manner (EC50 = 48.6 ± 1.8 nm) (Fig. 2b), with a maximal increase of 6.5-fold, comparable with that induced by the β1-AR agonist norepinephrine (NE at 10−7m) (37.9 ± 4.7 and 36.7 ± 7.7 in arbitrary units, respectively). Fig. 3a shows the time courses of β2-AR effects on contraction amplitude and duration (t½) in both PTX-treated and non-treated cells. In addition to the 1.5-fold potentiation of the β2-AR inotropic response, inhibition of Gi function allowed zinterol to induce a de novo relaxant effect in rat cardiomyocytes. The β2-AR-induced lusitropic effect in PTX-treated cells is highly comparable with that of β1-AR stimulation in control cells (Fig. 3, a and b). Furthermore, the time course of the β2-AR-induced Ser16 PLB phosphorylation was tightly correlated to the time course of the relaxant effect in PTX-treated cardiomyocytes, both reaching a steady state within 5 min (Fig. 3c). In contrast, neither the β1-AR-mediated contractile nor its relaxant response was affected by PTX (Fig. 3b). These results strongly suggest that the β2-AR/Gi coupling functionally compartmentalizes the β2-AR/Gs-mediated cAMP signaling, altering the quality as well as the magnitude of its cellular response. To elucidate further the mechanism underlying the Gi-mediated spatial control of β2-AR signaling, we measured the PKA activity following β-AR subtype stimulation. Similar to β1-AR stimulation, β2-AR activation also significantly increased the PKA activity ratio in both soluble and particulate fractions (Fig. 4). This suggests that, unlike β1-AR, β2-AR-mediated increases in cAMP accumulation (8Xiao R.P. Hohl C. Altschuld R. Jones L. Livingston B. Ziman B. Tantini B. Lakatta E.G. J. Biol. Chem. 1994; 269: 19151-19156Abstract Full Text PDF PubMed Google Scholar, 10Zhou Y.Y. Cheng H. Bogdanov K.Y. Hohl C. Altschuld R. Lakatta E.G. Xiao R.P. Am. J. Physiol. 1997; 273: H1611-H1618PubMed Google Scholar) and PKA activation (Fig. 4a) are dissociated from Ser16 PLB phosphorylation (Fig. 2). Surprisingly, PTX did not significantly affect the response of PKA in either fraction (Fig. 4), suggesting that the cross-talk of Gs and Gi signaling following β2-AR stimulation may occur downstream of PKA (see "Discussion"). We therefore examined the potential involvement of protein phosphatases in β2-AR-mediated Gisignaling. Rat cardiomyocytes were treated with calyculin A (3 × 10−8m) for 20 min to inhibit protein phosphatases. Control experiments showed that calyculin A at this concentration had only a marginal effect on contraction amplitude (127.1 ± 18.6% of control, n = 4), but induced a time- and dose-dependent augmentation in contraction at higher concentrations (data not shown). Interestingly, preincubation of cells with calyculin A at this near-threshold concentration markedly and selectively potentiated the submaximal contractile response to the β2-AR agonist zinterol (10−6m), whereas it had no effect on the submaximal β1-AR (NE, 10−8m)-stimulated contractile response (Fig. 5). Thus, the effects of protein phosphatase inhibition are similar to that of Gi inhibition by PTX, enhancing the contractile response in a β2-AR-specific manner (Fig. 3). This result strongly suggests that protein phosphatases are likely to be involved in the β2-AR/Gi-directed signaling. This conclusion was further substantiated by the observation that calyculin A failed to potentiate further the β2-AR-mediated contractile response if the Gi pathway is disrupted by PTX treatment (Fig. 5). Therefore, protein phosphatases may serve as a key element of the β2-AR-coupled Gi signaling cascade to spatially control the Gs-mediated cAMP/PKA signaling.Figure 5Involvement of protein phosphatases in the localization of β2-AR signaling. The protein phosphatase inhibitor, calyculin A (CalA, 3 × 10−8m), selectively potentiates the β2-AR agonist zinterol (Zin, 10−6m)-induced inotropic effect but has no further potentiating effect on the β2-AR contractile response in the presence of PTX pretreatment. The data (mean ± S.E., n = 7–19, *p < 0.05 versus zinterol) are expressed as percent change from values recorded before the application of the β-AR agonist. Base-line contractility is 5.98 ± 0.38 (n = 37), 5.20 ± 0.41 (n = 21), 5.04 ± 0.26 (n = 7), and 4.28 ± 0.48% (n = 7) of cell rest length for untreated, calyculin A-treated, PTX-treated, and PTX plus calyculin A-treated cells, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Recent advances in β2-AR signaling have provided evidence for a novel subcellular compartmentalization of cAMP signaling. Specifically, although both β1- and β2-AR stimulation enhance cAMP accumulation (8Xiao R.P. Hohl C. Altschuld R. Jones L. Livingston B. Ziman B. Tantini B. Lakatta E.G. J. Biol. Chem. 1994; 269: 19151-19156Abstract Full Text PDF PubMed Google Scholar, 10Zhou Y.Y. Cheng H. Bogdanov K.Y. Hohl C. Altschuld R. Lakatta E.G. Xiao R.P. Am. J. Physiol. 1997; 273: H1611-H1618PubMed Google Scholar) and PKA activity and modulate ICa via cAMP/PKA-dependent signaling, the β2-AR stimulation is uncoupled from the phosphorylation of more remote proteins (8Xiao R.P. Hohl C. Altschuld R. Jones L. Livingston B. Ziman B. Tantini B. Lakatta E.G. J. Biol. Chem. 1994; 269: 19151-19156Abstract Full Text PDF PubMed Google Scholar, 9Altschuld R.A. Starling R.C. Hamlin R.L. Billman G.E. Hensley J. Castillo L. Fertel R.H. Hohl C.M. Robitaille P.M. Jones L.R. Xiao R.P. Lakatta E.G. Circulation. 1995; 92: 1612-1618Crossref PubMed Scopus (126) Google Scholar, 11Kuschel M. Zhou Y.Y. Spurgeon H.A. Bartel S. Karczewski P. Zhang S.J. Krause E.G. Lakatta E.G. Xiao R.P. Circulation. 1999; 99: 2458-2465Crossref PubMed Scopus (106) Google Scholar). This indicates that the signaling may be highly localized to sarcolemmal microdomains or that it can be transmitted to cytoplasmic sites but locally inactivated there. The key question then is what mechanism enforces the tight spatial control of β2-AR-mediated cAMP signaling. In principle, a localized cAMP signaling could arise from localization of signaling components,e.g. localization of cAMP by phosphodiesterases (22Jurevicius J. Fischmeister R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 295-299Crossref PubMed Scopus (311) Google Scholar, 23Hohl C.M. Li Q.A. Circ. Res. 1991; 69: 1369-1379Crossref PubMed Scopus (108) Google Scholar) or PKA by specific anchoring proteins of PKA (24Dell'Acqua M.L. Scott J.D. J. Biol. Chem. 1997; 272: 12881-12884Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 25Mochly-Rosen D. Science. 1995; 268: 247-251Crossref PubMed Scopus (835) Google Scholar). A close spatial association of Ca2+ channels with adenylyl cyclase and PKA (26Gray P.C. Johnson B.D. Westenbroek R.E. Hayes L.G. Yates J.R. Scheuer T. Catterall W.A. Murphy B.J. Neuron. 1998; 20: 1017-1026Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 27Gao T. Puri T.S. Gerhardstein B.L. Chien A.J. Green R.D. Hosey M.M. J. Biol. Chem. 1997; 272: 19401-19407Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar) could provide a structural basis for the localized cAMP-dependent modulation of l-type Ca2+ channels following β2-AR stimulation. However, since both cAMP and catalytic subunits of PKA (released following activation) are diffusive molecules, additional mechanisms should be involved to restrict actively their signaling to certain subcellular domains. In the present study, we demonstrated that, apart from localization of signaling molecules of the cAMP/PKA cascade (22Jurevicius J. Fischmeister R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 295-299Crossref PubMed Scopus (311) Google Scholar, 23Hohl C.M. Li Q.A. Circ. Res. 1991; 69: 1369-1379Crossref PubMed Scopus (108) Google Scholar, 24Dell'Acqua M.L. Scott J.D. J. Biol. Chem. 1997; 272: 12881-12884Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 25Mochly-Rosen D. Science. 1995; 268: 247-251Crossref PubMed Scopus (835) Google Scholar), an interaction between functionally opposing signal transduction pathways can also create compartmentalization of receptor-mediated signaling. In particular, following inhibition of Gi function by PTX treatment, β2-AR stimulation markedly increased Ser16 PLB phosphorylation and elicited a de novolusitropic effect in rat ventricular myocytes, which is highly comparable with that following β1-AR stimulation. Thus, inhibition of Gi proteins converts the β2-AR signaling mode to that of β1-AR-like signaling. These results strongly suggest that the β2-AR/Gicoupling effectively compartmentalizes the β2-AR/Gs-mediated cAMP signaling, altering not only the magnitude but also the quality of its cardiac response. It is well established that activation of protein phosphatases functionally counterbalances cellular effects of protein kinases. Recently, it has been reported that the inhibitory effect of the Gi protein-coupled muscarinic receptor stimulation on the β-AR-induced cAMP signaling is largely mediated by activation of protein phosphatases (28Herzig S. Meier A. Pfeiffer M. Neumann J. Pfluegers Arch. 1995; 429: 531-538Crossref PubMed Scopus (57) Google Scholar). Furthermore, angiotensin II increases protein phosphatase 2A activity in cultured neurons through a Gi protein-dependent mechanism (29Huang X.C. Richards E.M. Sumners C. J. Neurochem. 1995; 65: 2131-2137Crossref PubMed Scopus (57) Google Scholar). We therefore investigated the potential involvement of protein phosphatases in the cross-talk of the β2-AR-stimulated Gs/Gi signaling. Interestingly, calyculin A, a non-selective protein phosphatase inhibitor, mimics the PTX effect. Both interventions, Gi inhibition by PTX or protein phosphatase inhibition by calyculin A, had a non-additive potentiating effect on β2-AR-mediated contractile response when applied together, suggesting that PTX and calyculin A act on a common signaling pathway. Therefore, the negating and spatial restricting effects of β2-AR-activated Gi proteins on the Gs-directed signaling might be mediated by a modulation of the protein phosphatase/kinase balance. For instance, a high dephosphorylation rate of non-sarcolemmal proteins following β2-AR stimulation might negate the PKA-mediated phosphorylation of PLB (and other cytoplasmic proteins). β2-AR-mediated Gi protein activation could either directly modulate protein phosphatase activity or inhibit PKA activation at subcellular compartments and secondarily modulate the protein phosphatase inhibitor 1 and therefore protein phosphatase activity (7Neumann J. Gupta R.C. Schmitz W. Scholz H. Nairn A.C. Watanabe A.M. Circ. Res. 1991; 69: 1450-1457Crossref PubMed Scopus (86) Google Scholar). In other words, the Gi protein activation could negate the β2-AR/Gs-mediated PKA activation in certain subcellular microdomains, resulting in a higher protein phosphatase activity and a lower protein phosphorylation in those subcellular microdomains, which is functionally indistinguishable from a Gi-mediated direct activation of protein phosphatases. Previous studies in frog and canine hearts have provided evidence for the involvement of phosphodiesterases in the compartmentalization of β-AR signaling (22Jurevicius J. Fischmeister R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 295-299Crossref PubMed Scopus (311) Google Scholar, 23Hohl C.M. Li Q.A. Circ. Res. 1991; 69: 1369-1379Crossref PubMed Scopus (108) Google Scholar). However, our previous studies have demonstrated that PTX treatment has no significant effect on β2-AR-mediated global cAMP accumulation (10Zhou Y.Y. Cheng H. Bogdanov K.Y. Hohl C. Altschuld R. Lakatta E.G. Xiao R.P. Am. J. Physiol. 1997; 273: H1611-H1618PubMed Google Scholar). The present results show further that β2-AR-stimulated increase in PKA activity in two different subcellular fractions is insensitive to PTX. Although the non-additive effect of protein phosphatase and Gi protein inhibition suggests, but does not prove, that β2-AR stimulation is modulating protein phosphatase activity through a PKA-independent pathway, these standard measures of global cAMP levels and PKA activity in cardiomyocytes provide no insight into the highly localized signaling. Although we are unable to distinguish if β2-AR/Gi signaling modulates protein phosphatases PKA-independent or -dependent due to technical limitation, the present results strongly suggest that protein phosphatases are critically involved in the β2-AR/Gi signaling, contributing to the functional compartmentalization of the β2-AR/Gs signaling in rat ventricular myocytes. It is noteworthy that the β2-AR-mediated cardiac response and the extent of the Gi protein coupling may vary substantially among species, resulting in an enormous diversity in cardiac β2-AR-mediated responses and its sensitivity to PTX. In mouse cardiomyocytes β2-ARs are not functional unless Gi function is inhibited by PTX treatment, indicating a high level of Gi protein coupling (16Xiao R.P. Avdonin P. Zhou Y.Y. Cheng H. Akhter S.A. Eschenhagen T. Lefkowitz R.J. Koch W.J. Lakatta E.G. Circ. Res. 1999; 84: 43-52Crossref PubMed Scopus (337) Google Scholar). In contrast, β2-AR stimulation does induce positive inotropic and lusitropic effects as well as phosphorylation of regulatory proteins in the failing human heart (30Kaumann A.J. Sanders L. Lynham J.A. Bartel S. Kuschel M. Karczewski P. Krause E.G. Mol. Cell. Biochem. 1996; 163–164: 113-123Crossref PubMed Scopus (51) Google Scholar, 31Kaumann A.J. Bartel S. Molenaar P. Sanders L. Burrell K. Vetter D. Hempel P. Karczewski P. Krause E.G. Circulation. 1999; 99: 65-72Crossref PubMed Scopus (117) Google Scholar). Between these extremes, β2-AR stimulation in rat cardiomyocytes induces significant increases in ICa and contractility in the absence of phosphorylation of cytoplasmic regulatory proteins. PTX treatment further enhances the β2-AR contractile response (17Xiao R.P. Ji X. Lakatta E.G. Mol. Pharmacol. 1995; 47: 322-329Crossref PubMed Scopus (201) Google Scholar) and restores its ability to phosphorylate cytoplasmic regulatory proteins in this species. The situation in canine myocytes is similar to that of rat myocytes, except that β2-AR does induce lusitropic as well as inotropic effects in the absence of cytoplasmic protein phosphorylation (9Altschuld R.A. Starling R.C. Hamlin R.L. Billman G.E. Hensley J. Castillo L. Fertel R.H. Hohl C.M. Robitaille P.M. Jones L.R. Xiao R.P. Lakatta E.G. Circulation. 1995; 92: 1612-1618Crossref PubMed Scopus (126) Google Scholar, 11Kuschel M. Zhou Y.Y. Spurgeon H.A. Bartel S. Karczewski P. Zhang S.J. Krause E.G. Lakatta E.G. Xiao R.P. Circulation. 1999; 99: 2458-2465Crossref PubMed Scopus (106) Google Scholar). The aforementioned data also illustrate that the same signaling molecule, cAMP, mediates remarkably different cardiac functional responses following β1- and β2-AR stimulation (8Xiao R.P. Hohl C. Altschuld R. Jones L. Livingston B. Ziman B. Tantini B. Lakatta E.G. J. Biol. Chem. 1994; 269: 19151-19156Abstract Full Text PDF PubMed Google Scholar, 9Altschuld R.A. Starling R.C. Hamlin R.L. Billman G.E. Hensley J. Castillo L. Fertel R.H. Hohl C.M. Robitaille P.M. Jones L.R. Xiao R.P. Lakatta E.G. Circulation. 1995; 92: 1612-1618Crossref PubMed Scopus (126) Google Scholar, 10Zhou Y.Y. Cheng H. Bogdanov K.Y. Hohl C. Altschuld R. Lakatta E.G. Xiao R.P. Am. J. Physiol. 1997; 273: H1611-H1618PubMed Google Scholar, 11Kuschel M. Zhou Y.Y. Spurgeon H.A. Bartel S. Karczewski P. Zhang S.J. Krause E.G. Lakatta E.G. Xiao R.P. Circulation. 1999; 99: 2458-2465Crossref PubMed Scopus (106) Google Scholar, 12Xiao R.P. Lakatta E.G. Circ. Res. 1993; 73: 286-300Crossref PubMed Scopus (210) Google Scholar). Analogously, it has been shown that intracellular Ca2+ located in different subcellular compartments may mediate distinctly different and sometimes even opposing cellular functions. For instance, a global elevation in cytosolic Ca2+ in arterial smooth muscle cells causes vasoconstriction, but Ca2+ sparks near the sarcolemma induce relaxation (32Nelson M.T. Cheng H. Rubart M. Santana L.F. Bonev A.D. Knot H.J. Lederer W.J. Science. 1995; 270: 633-637Crossref PubMed Scopus (1220) Google Scholar). In this case, local Ca2+ gradients are possible, because various endogenous binding sites buffer Ca2+ of discrete origins. Thus, physical or functional compartmentalization of ubiquitous intracellular messengers, such as cAMP and Ca2+, creates specificity and diversity of a given receptor-mediated signaling. In summary, we have demonstrated that, in addition to the potentiation of the inotropic response, inhibition of Gi function by PTX induces a de novo lusitropic effect and PLB phosphorylation following β2-AR stimulation in rat ventricular myocytes. These results suggest a contribution of β2-AR/Gi-coupled signaling to the compartmentalization of β2-AR/Gs-stimulated cAMP/PKA signaling, possibly through a protein phosphatase-dependent mechanism. In addition, the present study demonstrates that compartmentalization of a common second messenger-directed signaling allows for selective modulation of a variety of target proteins and cellular processes, creating signaling specificity and versatility among closely related G protein-coupled receptors.
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