Cytoplasmic cAMP concentrations in intact cardiac myocytes

In cardiac myocytes there is evidence that activation of some receptors can regulate protein kinase A (PKA)-dependent responses by stimulating cAMP production that is limited to discrete intracellular domains. We previously developed a computational model of compartmentalized cAMP signaling to investigate the feasibility of this idea. The model was able to reproduce experimental results demonstrating that both beta(1)-adrenergic and M(2) muscarinic receptor-mediated cAMP changes occur in microdomains associated with PKA signaling. However, the model also suggested that the cAMP concentration throughout most of the cell could be significantly higher than that found in PKA-signaling domains. In the present study we tested this counterintuitive hypothesis using a freely diffusible fluorescence resonance energy transfer-based biosensor constructed from the type 2 exchange protein activated by cAMP (Epac2-camps). It was determined that in adult ventricular myocytes the basal cAMP concentration detected by the probe is approximately 1.2 muM, which is high enough to maximally activate PKA. Furthermore, the probe detected responses produced by both beta(1) and M(2) receptor activation. Modeling suggests that responses detected by Epac2-camps mainly reflect what is happening in a bulk cytosolic compartment with little contribution from microdomains where PKA signaling occurs. These results support the conclusion that even though beta(1) and M(2) receptor activation can produce global changes in cAMP, compartmentation plays an important role by maintaining microdomains where cAMP levels are significantly below that found throughout most of the cell. This allows receptor stimulation to regulate cAMP activity over concentration ranges appropriate for modulating both higher (e.g., PKA) and lower affinity (e.g., Epac) effectors.     

Differential Association of Phosphodiesterase 4D Isoforms with ��2-Adrenoceptor in Cardiac Myocytes

cAMP and protein kinase A (PKA) activation represents a key signaling mechanism upon β-adrenergic stimulation under stress. Both β₁- and β₂-adrenoreceptor (ARs) subtypes induce cAMP accumulation, yet play distinct roles in cardiac contraction and myocyte apoptosis. Differences in controlling cAMP/PKA activities through the assembly of complexes between the receptors and cAMP-specific phosphodiesterases contribute to the distinct biological outcomes. Here, we demonstrate that β₂ARs form signaling complexes with a set of PDE4D isoforms expressed in cardiac myocytes. PDE4D9 and PDE4D8 bind to the β₂AR at resting conditions; however, agonist stimulation induces dissociation of PDE4D9 from the receptor but recruitment of PDE4D8 to the receptor. Agonist stimulation also induces recruitment of PDE4D5 to the β₂AR. Moreover, the receptor-associated PDE4D isoforms play distinct roles in controlling cAMP activities and regulating the PKA phosphorylation of the receptor and myocyte contraction rate responses. Knockdown of PDE4D9 with short hairpin RNA enhances the β₂AR-induced cAMP signaling, whereas knockdown of PDE4D8 only slightly prolongs the receptor-induced cAMP signaling in myocytes. Inhibition of PDE4D9 and PDE4D5 enhances the base-line levels of contraction rates, whereas inhibition of PDE4D9 and PDE4D8 enhances the maximal contraction rate increases upon activation of β₂AR. Our data underscore the complex regulation of intracellular cAMP by β₂AR-associated phosphodiesterase enzymes to enforce the specificity of the receptor signaling for physiological responses.     

Arrestin times for compartmentalised cAMP signalling and phosphodiesterase-4 enzymes

Various methods reveal that cyclic AMP (cAMP) signalling in cells is compartmentalised. These methods use FRET probes based upon either protein kinase A (PKA) or EPAC, cAMP-gated ion channels, or the selective activation of AKAP-anchored PKA isoforms. The basis of compartmentalisation involves point sources of cAMP generation within sub-domains of the plasma membrane coupled to degradation by spatially segregated, anchored forms of cAMP phosphodiesterases. cAMP-specific phosphodiesterase-4 (PDE4) isoforms play a central role in determining compartmentalisation, as exemplified in cardiac myocytes and T cells. The PKA phosphorylation status of the beta2-adrenoreceptor, and hence its ability to switch its signalling from G(s) to G(i) and thus to activate ERK, is regulated dynamically by the agonist-stimulated recruitment of PDE4 to the receptor in complex with beta-arrestin. The co-receptor CD28 enhances signalling through the T-cell receptor by recruiting a PDE4/beta-arrestin complex, which then attenuates PKA phosphorylation of Csk.     

A Long Lasting β1 Adrenergic Receptor Stimulation of cAMP/Protein Kinase A (PKA) Signal in Cardiac Myocytes

Small-molecule, ligand-activated G protein-coupled receptors are generally thought to be rapidly desensitized within a period of minutes through receptor phosphorylation and internalization after repeated or prolonged stimulation. This transient G protein-coupled receptor activation remains at odds with many observed long-lasting cellular and physiological responses. Here, using live cell imaging of cAMP with a FRET-based biosensor and myocyte contraction assay, we show that the catecholamine-activated β₁ adrenergic receptor (β₁AR) continuously stimulates second messenger cAMP synthesis in primary cardiac myocytes and neurons, which lasts for more than 8 h (a decay t_½ of 3.9 h) in cardiac myocytes. However, the β₁AR-induced cAMP signal is counterbalanced and masked by the receptor-bound phosphodiesterase (PDE) 4D8-dependent cAMP hydrolysis. Inhibition of PDE4 activity recovers the receptor-induced cAMP signal and promotes contractile response in mouse hearts during extended periods of agonist stimulation. β₁AR associates with PDE4D8 through the receptor C-terminal PDZ motif-dependent binding to synaptic-associated protein 97 (SAP97). Knockdown of SAP97 or mutation of the β₁AR PDZ motif disrupts the complex and promotes sustained agonist-induced cAMP activity, PKA phosphorylation, and cardiac myocyte contraction response. Together, these findings unveil a long lasting adrenergic signal in neurons and myocytes under prolonged stimulation and an underappreciated role of PDE that is essential in classic receptor signaling desensitization and in maintaining a long lasting cAMP equilibrium for ligand-induced physiological response.     

Receptor number and caveolar co-localization determine receptor coupling efficiency to adenylyl cyclase

Recent evidence suggests that many signaling molecules localize in microdomains of the plasma membrane, particularly caveolae. In this study, overexpression of adenylyl cyclase was used as a functional probe of G protein-coupled receptor (GPCR) compartmentation. We found that three endogenous receptors in neonatal rat cardiomyocytes couple with different levels of efficiency to the activation of adenylyl cyclase type 6 (AC6), which localizes to caveolin-rich membrane fractions. Overexpression of AC6 enhanced the maximal cAMP response to beta(1)-adrenergic receptor (beta(1)AR)-selective activation 3.7-fold, to beta(2)AR-selective activation only 1.6-fold and to prostaglandin E(2) (PGE(2)) not at all. Therefore, the rank order of efficacy in coupling to AC6 is beta(1)AR > beta(2)AR > prostaglandin E(2) receptor (EP(2)R). beta(2)AR coupling efficiency was greater when we overexpressed the receptor or blocked its desensitization by expressing betaARKct, an inhibitor of G protein-coupled receptor kinase activation, but was not significantly greater when cells were treated with pertussis toxin. Assessment of receptor and AC expression indicated co-localization of AC5/6, beta(1)AR, and beta(2)AR, but not EP(2)R, in caveolin-rich membranes and caveolin-3 immunoprecipitates, likely explaining the observed activation of AC6 by betaAR subtypes but lack thereof by PGE(2). When cardiomyocytes were stimulated with a betaAR agonist, beta(2)AR were no longer found in caveolin-3 immunoprecipitates; an effect that was blocked by expression of betaARKct. Thus, agonist-induced translocation of beta(2)AR out of caveolae causes a sequestration of receptor from effector and likely contributes to the lower efficacy of beta(2)AR coupling to AC6 as compared with beta(1)AR, which do not similarly translocate. Therefore, spatial co-localization is a key determinant of efficiency of coupling by particular extracellular signals to activation of GPCR-linked effectors.     

The beta3-adrenergic receptor activates mitogen-activated protein kinase in adipocytes through a Gi-dependent mechanism

Promiscuous coupling between G protein-coupled receptors and multiple species of heterotrimeric G proteins provides a potential mechanism for expanding the diversity of G protein-coupled receptor signaling. We have examined the mechanism and functional consequences of dual Gs/Gi protein coupling of the beta3-adrenergic receptor (beta3AR) in 3T3-F442A adipocytes. The beta3AR selective agonist disodium (R, R)-5-[2[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]propyl]-1, 3-benzodioxole-2,2-dicarboxylate (CL316,243) stimulated a dose-dependent increase in cAMP production in adipocyte plasma membrane preparations, and pretreatment of cells with pertussis toxin resulted in a further 2-fold increase in cAMP production by CL316,243. CL316,243 (5 microM) stimulated the incorporation of 8-azido-[32P]GTP into Galphas (1.57 +/- 0.12; n = 3) and Galphai (1. 68 +/- 0.13; n = 4) in adipocyte plasma membranes, directly demonstrating that beta3AR stimulation results in Gi-GTP exchange. The beta3AR-stimulated increase in 8-azido-[32P]GTP labeling of Galphai was equivalent to that obtained with the A1-adenosine receptor agonist N6-cyclopentyladenosine (1.56 +/- 0.07; n = 4), whereas inclusion of unlabeled GTP (100 microM) eliminated all binding. Stimulation of the beta3AR in 3T3-F442A adipocytes led to a 2-3-fold activation of mitogen-activated protein (MAP) kinase, as measured by extracellular signal-regulated kinase-1 and -2 (ERK1/2) phosphorylation. Pretreatment of cells with pertussis toxin (PTX) eliminated MAP kinase activation by beta3AR, demonstrating that this response required receptor coupling to Gi. Expression of the human beta3AR in HEK-293 cells reconstituted the PTX-sensitive stimulation of MAP kinase, demonstrating that this phenomenon is not exclusive to adipocytes or to the rodent beta3AR. ERK1/2 activation by the beta3AR was insensitive to the cAMP-dependent protein kinase inhibitor H-89 but was abolished by genistein and AG1478. These data indicate that constitutive beta3AR coupling to Gi proteins serves both to restrain Gs-mediated activation of adenylyl cyclase and to initiate additional signal transduction pathways, including the ERK1/2 MAP kinase cascade.     

S1P1 receptor localization confers selectivity for Gi-mediated cAMP and contractile responses

Adult mouse ventricular myocytes express S1P(1), S1P(2), and S1P(3) receptors. S1P activates Akt and ERK in adult mouse ventricular myocytes through a pertussis toxin-sensitive (G(i/o)-mediated) pathway. Akt and ERK activation by S1P are reduced approximately 30% in S1P(3) and 60% in S1P(2) receptor knock-out myocytes. With combined S1P(2,3) receptor deletion, activation of Akt is abolished and ERK activation is reduced by nearly 90%. Thus the S1P(1) receptor, while present in S1P(2,3) receptor knock-out myocytes, is unable to mediate Akt or ERK activation. In contrast, S1P induces pertussis toxin-sensitive inhibition of isoproterenol-stimulated cAMP accumulation in both WT and S1P(2,3) receptor knock-out myocytes demonstrating that the S1P(1) receptor can functionally couple to G(i). An S1P(1) receptor selective agonist, SEW2871, also decreased cAMP accumulation but failed to activate ERK or Akt. To determine whether localization of the S1P(1) receptor mediates this signaling specificity, methyl-beta-cyclodextrin (MbetaCD) treatment was used to disrupt caveolae. The S1P(1) receptor was concentrated in caveolar fractions, and associated with caveolin-3 and this localization was disrupted by MbetaCD. S1P-mediated activation of ERK or Akt was not diminished but inhibition of cAMP accumulation by S1P and SEW2871 was abolished by MbetaCD treatment. S1P inhibits the positive inotropic response to isoproterenol and this response is also mediated through the S1P(1) receptor and lost following caveolar disruption. Thus localization of S1P(1) receptors to caveolae is required for the ability of this receptor to inhibit adenylyl cyclase and contractility but compromises receptor coupling to Akt and ERK.     

Adenosine A2A and beta-adrenergic calcium transient and contractile responses in rat ventricular myocytes

The adenosine A2A receptor (A2AR) enhances cardiac contractility, and the adenosine A1R receptor (A1R) is antiadrenergic by reducing the adrenergic beta1 receptor (beta1R)-elicited increase in contractility. In this study we compared the A2AR-, A1R-, and beta1R-elicited actions on isolated rat ventricular myocytes in terms of Ca transient and contractile responses involving PKA and PKC. Stimulation of A2AR with 2 microM (approximately EC50) CGS-21680 (CGS) produced a 17-28% increase in the Ca transient ratio (CTR) and maximum velocities (Vmax) of transient ratio increase (+MVT) and recovery (-MVT) but no change in the time-to-50% recovery (TTR). CGS increased myocyte sarcomere shortening (MSS) and the maximum velocities of shortening (+MVS) and relaxation (-MVS) by 31-34% with no change in time-to-50% relengthening (TTL). beta1R stimulation using 2 nM (approximately EC50) isoproterenol (Iso) increased CTR, +MVT, and -MVT by 67-162% and decreased TTR by 43%. Iso increased MSS, +MVS, and -MVS by 153-174% and decreased TTL by 31%. The A2AR and beta1R Ca transient and contractile responses were not additive. The PKA inhibitor Rp-adenosine 3',5'-cyclic monophosphorothioate triethylamonium salt prevented both the CGS- and Iso-elicited contractile responses. The PKC inhibitors chelerythrine and KIE1-1 peptide (PKCepsilon specific) prevented the antiadrenergic action of A1R but did not influence A2AR-mediated increases in contractile variables. The findings suggest that cardiac A2AR utilize cAMP/PKA like beta1R, but the Ca transient and contractile responses are less in magnitude and not equally affected. Although PKC is important in the A1R antiadrenergic action, it does not seem to play a role in A2AR-elicited Ca transient and contractile events.     

Caveolar localization dictates physiologic signaling of beta 2-adrenoceptors in neonatal cardiac myocytes

There is a growing body of evidence that G protein-coupled receptors function in the context of plasma membrane signaling compartments. These compartments may facilitate interaction between receptors and specific downstream signaling components while restricting access to other signaling molecules. We recently reported that beta(1)- and beta(2)-adrenergic receptors (AR) regulate the intrinsic contraction rate in neonatal mouse myocytes through distinct signaling pathways. By studying neonatal myocytes isolated from beta(1)AR and beta(2)AR knockout mice, we found that stimulation of the beta(1)AR leads to a protein kinase A-dependent increase in the contraction rate. In contrast, stimulation of the beta(2)AR has a biphasic effect on the contraction rate. The biphasic effect includes an initial protein kinase A-independent increase in the contraction rate followed by a sustained decrease in the contraction rate that can be blocked by pertussis toxin. Here we present evidence that caveolar localization is required for physiologic signaling by the beta(2)AR but not the beta(1)AR in neonatal cardiac myocytes. Evidence for beta(2)AR localization to caveolae includes co-localization by confocal imaging, co-immunoprecipitation of the beta(2)AR and caveolin 3, and co-migration of the beta(2)AR with a caveolin-3-enriched membrane fraction. The beta(2)AR-stimulated increase in the myocyte contraction rate is increased by approximately 2-fold and markedly prolonged by filipin, an agent that disrupts lipid rafts such as caveolae and significantly reduces co-immunoprecipitation of beta(2)AR and caveolin 3 and co-migration of beta(2)AR and caveolin-3 enriched membranes. In contrast, filipin has no effect on beta(1)AR signaling. These observations suggest that beta(2)ARs are normally restricted to caveolae in myocyte membranes and that this localization is essential for physiologic signaling of this receptor subtype.     

Inhibition of spontaneous beta 2-adrenergic activation rescues beta 1-adrenergic contractile response in cardiomyocytes overexpressing beta 2-adrenoceptor

Cardiac-specific overexpression of the human beta(2)-adrenergic receptor (AR) in transgenic mice (TG4) enhances basal cardiac function due to ligand-independent spontaneous beta(2)-AR activation. However, agonist-mediated stimulation of either beta(1)-AR or beta(2)-AR fails to further enhance contractility in TG4 ventricular myocytes. Although the lack of beta(2)-AR response has been ascribed to an efficient coupling of the receptor to pertussis toxin-sensitive G(i) proteins in addition to G(s), the contractile response to beta(1)-AR stimulation by norepinephrine and an alpha(1)-adrenergic antagonist prazosin is not restored by pertussis toxin treatment despite a G(i) protein elevation of 1.7-fold in TG4 hearts. Since beta-adrenergic receptor kinase, betaARK1, activity remains unaltered, the unresponsiveness of beta(1)-AR is not caused by betaARK1-mediated receptor desensitization. In contrast, pre-incubation of cells with anti-adrenergic reagents such as muscarinic receptor agonist, carbachol (10(-5)m), or a beta(2)-AR inverse agonist, ICI 118,551 (5 x 10(-7)m), to abolish spontaneous beta(2)-AR signaling, both reduce the base-line cAMP and contractility and, surprisingly, restore the beta(1)-AR contractile response. The "rescued" contractile response is completely reversed by a beta(1)-AR antagonist, CGP 20712A. Furthermore, these results from the transgenic animals are corroborated by in vitro acute gene manipulation in cultured wild type adult mouse ventricular myocytes. Adenovirus-directed overexpression of the human beta(2)-AR results in elevated base-line cAMP and contraction associated with a marked attenuation of beta(1)-AR response; carbachol pretreatment fully revives the diminished beta(1)-AR contractile response. Thus, we conclude that constitutive beta(2)-AR activation induces a heterologous desensitization of beta(1)-ARs independent of betaARK1 and G(i) proteins; suppression of the constitutive beta(2)-AR signaling by either a beta(2)-AR inverse agonist or stimulation of the muscarinic receptor rescues the beta(1)-ARs from desensitization, permitting agonist-induced contractile response.     

Critical role of PDE4D in beta2-adrenoceptor-dependent cAMP signaling in mouse embryonic fibroblasts

One of the defining properties of beta2-adrenergic receptor (beta(2)AR) signaling is the transient and rapidly reversed accumulation of cAMP. Here we have investigated the contribution of different PDE4 proteins to the generation of this transient response. To this aim, mouse embryonic fibroblasts deficient in PDE4A, PDE4B, or PDE4D were generated, and the regulation of PDE activity, the accumulation of cAMP, and CREB phosphorylation in response to isoproterenol were monitored. Ablation of PDE4D, but not PDE4A or PDE4B, had a major effect on the beta-agonist-induced PDE activation, with only a minimal increase in PDE activity being retained in PDE4D knock-out (KO) cells. Accumulation of cAMP was markedly enhanced, and the kinetics of cAMP accumulation were altered in their properties in PDE4DKO but not PDE4BKO cells. Modest effects were observed in PDE4AKO mouse embryonic fibroblasts. The return to basal levels of both cAMP accumulation and CREB phosphorylation was greatly delayed in the PDE4DKO cells, suggesting that PDE4D is critical for dissipation of the beta2AR stimulus. This effect of PDE4D ablation was in large part due to inactivation of a negative feedback mechanism consisting of the PKA-mediated activation of PDE4D in response to elevated cAMP levels, as indicated by experiments using the cAMP-dependent protein kinase inhibitors H89 and PKI. Finally, PDE4D ablation affected the kinetics of beta2AR desensitization as well as the interaction of the receptor with Galphai. These findings demonstrate that PDE4D plays a major role in shaping the beta2AR signal.     

心脏中不同β肾上腺素受体亚型的信号体系及其病理生理意义

生理情况下,β肾上腺素受体(βAR)对心肌收缩和舒张活动起至关重要的作用;病理情况下,长期激动βAR可以诱发心肌细胞肥大、凋亡以及细胞坏死等心肌重塑性活动,从而参与了慢性心衰的发病过程。近十年以来,许多资料表明β1和β2肾上腺素受体亚型(β1AR和β2AR)共存于心脏中,且激动不同信号系统。短时间激动β1AR,使Gs蛋白-腺苷酸环化酶-环苷腺酸-蛋白激酶A(Gs-adenyly cyclase-cAMP-PKA)信号体系激活并广布于细胞内,而激动βAR则同时激活G1蛋白而产生空间及功能局限的cAMP信号;长时间激动β1AR和β2AR则对心肌细胞的命运产生不同影响：β1AR诱导细胞肥大和凋亡,β2AR促使细胞存活。β2AR的心肌保护作用是通过激活Gi蛋白-Gβγ-PI3K-Akt途径介导。但出乎意料,β1AR的心肌肥厚和凋亡效应并不依赖于经典的cAMP/PKA信号途径,而是激活钙,钙调素依赖性蛋白激酶Ⅱ(caMK Ⅱ)途径。用心肌特异性表达βAR亚型的转基因小鼠进行实验,进一步证实不同βAR亚型在调节心肌重塑和功能方面作用各异。βAR亚型作用不同的新观点不仅为β阻滞剂治疗慢性心衰提供了分子和细胞机制的依据,而且提出了选择性β1AR阻滞和β2AR激动联合治疗慢性心衰的新的治疗思路。     

Signaling from beta1- and beta2-adrenergic receptors is defined by differential interactions with PDE4

Beta1- and beta2-adrenergic receptors (betaARs) are highly homologous, yet they play clearly distinct roles in cardiac physiology and pathology. Myocyte contraction, for instance, is readily stimulated by beta1AR but not beta2AR signaling, and chronic stimulation of the two receptors has opposing effects on myocyte apoptosis and cell survival. Differences in the assembly of macromolecular signaling complexes may explain the distinct biological outcomes. Here, we demonstrate that beta1AR forms a signaling complex with a cAMP-specific phosphodiesterase (PDE) in a manner inherently different from a beta2AR/beta-arrestin/PDE complex reported previously. The beta1AR binds a PDE variant, PDE4D8, in a direct manner, and occupancy of the receptor by an agonist causes dissociation of this complex. Conversely, agonist binding to the beta2AR is a prerequisite for the recruitment of a complex consisting of beta-arrestin and the PDE4D variant, PDE4D5, to the receptor. We propose that the distinct modes of interaction with PDEs result in divergent cAMP signals in the vicinity of the two receptors, thus, providing an additional layer of complexity to enforce the specificity of beta1- and beta2-adrenoceptor signaling.     

Inhibition of human airway smooth muscle cell proliferation by beta 2-adrenergic receptors and cAMP is PKA independent: evidence for EPAC involvement

Mechanisms by which beta-adrenergic receptor (beta AR) agonists inhibit proliferation of human airway smooth muscle (HASM) cells were investigated because of their potential relevance to smooth muscle hyperplasia in asthma. We hypothesized that beta AR agonists would inhibit mitogenesis in HASM cells via the beta 2AR, an increase in cAMP, and PKA activation. HASM cells were treated for 24 h with various agents and then analyzed for [3H]thymidine incorporation as a measure of cell proliferation. EGF stimulated proliferation by approximately 10-fold. The nonselective beta AR agonist isoproterenol and the beta 2AR-selective agonists albuterol and salmeterol inhibited EGF-stimulated proliferation by more than 50%, with half-maximal effects at 4.8 nM, 110 nM, and 6.7 nM, respectively. A beta 2AR-selective antagonist inhibited the isoproterenol effect with 100-fold greater potency than a beta 1AR-selective antagonist, confirming beta 2AR involvement in the inhibition of proliferation. The cAMP-elevating agents PGE2 and forskolin decreased EGF-induced proliferation, suggesting cAMP as the mediator. beta 2AR agonists and forskolin also inhibited proliferation stimulated by lysophosphatidic acid (LPA) as well as the synergistic proliferation stimulated by LPA+EGF. Importantly, PKA-selective cAMP analogs did not inhibit proliferation at concentrations that maximally activated PKA (10-100 microM), whereas a cAMP analog selective for the exchange protein directly activated by cAMP (EPAC), 8-(4-chlorophenylthio)-2'-O-methyl-cAMP, maximally inhibited proliferation at a concentration that did not activate PKA (10 microM). These data show that beta 2AR agonists and other cAMP-elevating agents decrease proliferation in HASM cells via a PKA-independent mechanism, and they provide pharmacological evidence for involvement of EPAC or an EPAC-like cAMP effector protein instead.     

The PDZ binding motif of the beta 1 adrenergic receptor modulates receptor trafficking and signaling in cardiac myocytes

Beta(1) and beta(2) adrenergic receptors (AR) regulate the intrinsic contraction rate in neonatal mouse cardiac myocytes through distinct signaling pathways. It has been shown that stimulation of beta(1)ARs leads to a protein kinase A-dependent increase in contraction rate. In contrast, stimulation of beta(2)ARs has a biphasic effect on contraction rate, with an initial protein kinase A-independent increase followed by a sustained decrease that is blocked by pertussis toxin. The beta(2)AR undergoes agonist-induced endocytosis in cardiac myocytes while the beta(1)AR remains on the cell surface. It has been shown that a PDZ domain binding motif at the carboxyl terminus of beta(1)AR interacts with the postsynaptic density protein PSD-95 when both are expressed in HEK293 cells. We found that mutation of this PDZ binding motif in the beta(1)AR (beta(1)AR-PDZ) enabled agonist-induced internalization in cardiac myocytes. Moreover, stimulation of beta(1)AR-PDZ had a biphasic effect on the myocyte contraction rate similar to that observed following stimulation of the beta(2)AR. The secondary decrease in the contraction rate was mediated by G(i) and could be blocked by pertussis toxin. Furthermore, a non-selective endocytosis inhibitor, concanavalin A, inhibited the internalization of wild type beta(2)AR and the mutated beta(1)AR-PDZ, and blocked the coupling of both receptors to G(i). Finally, treating myocytes with a membrane-permeable peptide representing beta(1)AR PDZ motif caused the endogenous beta(1)AR to behave like beta(1)AR-PDZ. These studies suggest that association of the beta(1)AR with PSD-95 or a related protein dictates signaling specificity by retaining the receptor at the cell surface and preventing interaction with G(i).     

Cyclic nucleotide phosphodiesterase PDE1C1 in human cardiac myocytes

Isoforms in the PDE1 family of cyclic nucleotide phosphodiesterases were recently found to comprise a significant portion of the cGMP-inhibited cAMP hydrolytic activity in human hearts. We examined the expression of PDE1 isoforms in human myocardium, characterized their catalytic activity, and quantified their contribution to cAMP hydrolytic and cGMP hydrolytic activity in subcellular fractions of this tissue. Western blotting with isoform-selective anti-PDE1 monoclonal antibodies showed PDE1C1 to be the principal isoform expressed in human myocardium. Immunohistochemical analysis showed that PDE1C1 is distributed along the Z-lines and M-lines of cardiac myocytes in a striated pattern that differs from that of the other major dual-specificity cyclic nucleotide phosphodiesterase in human myocardium, PDE3A. Most of the PDE1C1 activity was recovered in soluble fractions of human myocardium. It binds both cAMP and cGMP with K(m) values of approximately 1 microm and hydrolyzes both substrates with similar catalytic rates. PDE1C1 activity in subcellular fractions was quantified using a new PDE1-selective inhibitor, IC295. At substrate concentrations of 0.1 microm, PDE1C1 constitutes the great majority of cAMP hydrolytic and cGMP hydrolytic activity in soluble fractions and the majority of cGMP hydrolytic activity in microsomal fractions, whereas PDE3 constitutes the majority of cAMP hydrolytic activity in microsomal fractions. These results indicate that PDE1C1 is expressed at high levels in human cardiac myocytes with an intracellular distribution distinct from that of PDE3A and that it may have a role in the integration of cGMP-, cAMP- and Ca(2+)-mediated signaling in these cells.     

Isoforms of cyclic nucleotide phosphodiesterase PDE3 and their contribution to cAMP hydrolytic activity in subcellular fractions of human myocardium

Three isoforms of PDE3 (cGMP-inhibited) cyclic nucleotide phosphodiesterase regulate cAMP content in different intracellular compartments of cardiac myocytes in response to different signals. We characterized the catalytic activity and inhibitor sensitivity of these isoforms by using recombinant proteins. We determined their contribution to cAMP hydrolysis in cytosolic and microsomal fractions of human myocardium at 0.1 and 1.0 microm cAMP in the absence and presence of Ca(2+)/calmodulin. We examined the effects of cGMP on cAMP hydrolysis in these fractions. PDE3A-136, PDE3A-118, and PDE3A-94 have similar K(m) and k(cat) values for cAMP and are equal in their sensitivities to inhibition by cGMP and cilostazol. In microsomes, PDE3A-136, PDE3A-118, and PDE3A-94 comprise the majority of cAMP hydrolytic activity under all conditions. In cytosolic fractions, PDE3A-118 and PDE3A-94 comprise >50% of the cAMP hydrolytic activity at 0.1 microm cAMP, in the absence of Ca(2+)/calmodulin. At 1.0 microm cAMP, in the presence of Ca(2+)/calmodulin, activation of Ca(2+)/calmodulin-activated (PDE1) and other non-PDE3 phosphodiesterases reduces their contribution to <20% of cAMP hydrolytic activity. cGMP inhibits cAMP hydrolysis in microsomal fractions by inhibiting PDE3 and in cytosolic fractions by inhibiting both PDE3 and PDE1. These findings indicate that the contribution of PDE3 isoforms to the regulation of cAMP hydrolysis in intracellular compartments of human myocardium and the effects of PDE3 inhibition on cAMP hydrolysis in these compartments are highly dependent on intracellular [Ca(2+)] and [cAMP], which are lower in failing hearts than in normal hearts. cGMP may amplify cAMP-mediated signaling in intracellular compartments of human myocardium by PDE3-dependent and PDE3-independent mechanisms.     

beta 2-adrenergic receptor activates extracellular signal-regulated kinases (ERKs) via the small G protein rap1 and the serine/threonine kinase B-Raf

G protein-coupled receptors can induce cellular proliferation by stimulating the mitogen-activated protein (MAP) kinase cascade. Heterotrimeric G proteins are composed of both alpha and betagamma subunits that can signal independently to diverse intracellular signaling pathways including those that activate MAP kinases. In this study, we examined the ability of isoproterenol, an agonist of the beta(2)-adrenergic receptor (beta(2)AR), to stimulate extracellular signal-regulated kinases (ERKs). Using HEK293 cells, which express endogenous beta(2)AR, we show that isoproterenol stimulates ERKs via beta(2)AR. This action of isoproterenol requires cAMP-dependent protein kinase and is insensitive to pertussis toxin, suggesting that Galpha(s) activation of cAMP-dependent protein kinase is required. Interestingly, beta(2)AR activates both the small G proteins Rap1 and Ras, but only Rap1 is capable of coupling to Raf isoforms. beta(2)AR inhibits the Ras-dependent activation of both Raf isoforms Raf-1 and B-Raf, whereas Rap1 activation by isoproterenol recruits and activates B-Raf. beta(2)AR activation of ERKs is not blocked by expression of RasN17, an interfering mutant of Ras, but is blocked by expression of either RapN17 or Rap1GAP1, both of which interfere with Rap1 signaling. We propose that isoproterenol can activate ERKs via Rap1 and B-Raf in these cells.     

Heterologous facilitation of G protein-activated K(+) channels by beta-adrenergic stimulation via cAMP-dependent protein kinase

To investigate possible effects of adrenergic stimulation on G protein-activated inwardly rectifying K(+) channels (GIRK), acetylcholine (ACh)-evoked K(+) current, I(KACh), was recorded from adult rat atrial cardiomyocytes using the whole cell patch clamp method and a fast perfusion system. The rise time of I(KACh ) was 0. 4 +/- 0.1 s. When isoproterenol (Iso) was applied simultaneously with ACh, an additional slow component (11.4 +/- 3.0 s) appeared, and the amplitude of the elicited I(KACh) was increased by 22.9 +/- 5.4%. Both the slow component of activation and the current increase caused by Iso were abolished by preincubation in 50 microM H89 (N-[2-((p -bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, a potent inhibitor of PKA). This heterologous facilitation of GIRK current by beta-adrenergic stimulation was further studied in Xenopus laevis oocytes coexpressing beta(2)-adrenergic receptors, m(2 )-receptors, and GIRK1/GIRK4 subunits. Both Iso and ACh elicited GIRK currents in these oocytes. Furthermore, Iso facilitated ACh currents in a way, similar to atrial cells. Cytosolic injection of 30-60 pmol cAMP, but not of Rp-cAMPS (a cAMP analogue that is inhibitory to PKA) mimicked the beta(2)-adrenergic effect. The possibility that the potentiation of GIRK currents was a result of the phosphorylation of the beta-adrenergic receptor (beta(2)AR) by PKA was excluded by using a mutant beta(2)AR in which the residues for PKA-mediated modulation were mutated. Overexpression of the alpha subunit of G proteins (Galpha(s)) led to an increase in basal as well as agonist-induced GIRK1/GIRK4 currents (inhibited by H89). At higher levels of expressed Galpha(s), GIRK currents were inhibited, presumably due to sequestration of the beta/gamma subunit dimer of G protein. GIRK1/GIRK5, GIRK1/GIRK2, and homomeric GIRK2 channels were also regulated by cAMP injections. Mutant GIRK1/GIRK4 channels in which the 40 COOH-terminal amino acids (which contain a strong PKA phosphorylation consensus site) were deleted were also modulated by cAMP injections. Hence, the structural determinant responsible is not located within this region. We conclude that, both in atrial myocytes and in Xenopus oocytes, beta-adrenergic stimulation potentiates the ACh-evoked GIRK channels via a pathway that involves PKA-catalyzed phosphorylation downstream from beta(2)AR.