The Arg389Gly ß1-adrenoceptor gene polymorphism influences the acute effects of ß-adrenoceptor blockade on contractility in the human heart.

BACKGROUND: The ß(1)-adrenoceptor (ß(1)AR) mediates cardiostimulatory effects of catecholamines in the heart. The Arg389Gly polymorphism of the ß(1)AR gene has recently been shown to determine the responsiveness to catecholamines in vitro, and we previously reported that dobutamine induced an augmented contractile response in humans homozygous for the Arg389 allele. The aim of the present study was to evaluate whether the Arg389Gly ß(1)AR gene polymorphism influences the responsiveness to ß-blocker treatment on cardiac contractility. METHODS: We investigated 30 healthy male volunteers who were genotyped for the Arg389Gly polymorphism and subjected to modified dobutamine stress-echocardiography (DSE) with and without pretreatment with metoprolol controlled release/extended release (CR/XL). Fractional shortening (FS) as a parameter for left-ventricular contractility and heart rate were measured. To control for pre- and afterload effects, rate corrected velocity of circumferential fiber shortening (Vcfc) was calculated. RESULTS: Homozygous Arg389 carriers had a significantly higher increase in contractility upon dobutamine stimulation than individuals carrying the Gly389 allele. Pretreatment with metoprolol CR/XL prior to DSE blunted the dobutamine-induced increase of FS and Vcfc in homozygous Arg389 carriers to the level of subjects carrying the Gly389 allele. In the latter group, metoprolol had a modest effect on the dobutamine-induced increase in FS at low concentrations of dobutamine, and no significant effect at peak stress (40 µg/kg/min of dobutamine). CONCLUSION: The responsiveness to ß-blockers with respect to cardiac contractility is determined by the Arg389Gly ß(1)AR gene polymorphism. These findings offer a molecular explanation for interindividual differences in the responsiveness to ß-blocker treatment in humans.

Phosphatidylinositol 3-kinase-dependent membrane recruitment of Rac-1 and p47phox is critical for alpha-platelet-derived growth factor receptor-induced production of reactive oxygen species.

Platelet-derived growth factor (PDGF) plays a critical role in the pathogenesis of proliferative diseases. NAD(P)H oxidase (Nox)-derived reactive oxygen species (ROS) are essential for signal transduction by growth factor receptors. Here we investigated the dependence of PDGF-AA-induced ROS production on the cytosolic Nox subunits Rac-1 and p47(phox), and we systematically evaluated the signal relay mechanisms by which the alphaPDGF receptor (alphaPDGFR) induces ROS liberation. Stimulation of the alphaPDGFR led to a time-dependent increase of intracellular ROS levels in fibroblasts. Pharmacological inhibitor experiments and enzyme activity assays disclosed Nox as the source of ROS. alphaPDGFR activation is rapidly followed by the translocation of p47(phox) and Rac-1 from the cytosol to the cell membrane. Experiments performed in p47(phox)(-/-) cells and inhibition of Rac-1 or overexpression of dominant-negative Rac revealed that these Nox subunits are required for PDGF-dependent Nox activation and ROS liberation. To evaluate the signaling pathway mediating PDGF-AA-dependent ROS production, we investigated Ph cells expressing mutant alphaPDGFRs that lack specific binding sites for alphaPDGFR-associated signaling molecules (Src, phosphatidylinositol 3-kinase (PI3K), phospholipase Cgamma, and SHP-2). Lack of PI3K signaling (but not Src, phospholipase Cgamma, or SHP-2) completely abolished PDGF-dependent p47(phox) and Rac-1 translocation, increase of Nox activity, and ROS production. Conversely, a mutant alphaPDGFR able to activate only PI3K was sufficient to mediate these subcellular events. Furthermore, the catalytic PI3K subunit p110alpha (but not p110beta) was identified as the crucial isoform that elicits alphaPDGFR-mediated production of ROS. Finally, bromodeoxyuridine incorporation and chemotaxis assays revealed that the lack of ROS liberation blunted PDGF-AA-dependent chemotaxis but not cell cycle progression. We conclude that PI3K/p110alpha mediates growth factor-dependent ROS production by recruiting p47(phox) and Rac-1 to the cell membrane, thereby assembling the active Nox complex. ROS are required for PDGF-AA-dependent chemotaxis but not proliferation.

PI3-kinase/Akt-dependent antiapoptotic signaling by the PDGF alpha receptor is negatively regulated by Src family kinases.

Regulation of growth factor dependent cell survival is crucial for development and disease progression. Here, we report a novel function of Src kinases as a negative regulator of platelet-derived growth factor (PDGF) dependent cell survival. We characterized a series of PDGF alpha receptor (PDGFRA) mutants, which lack the binding sites for Src, phosphatidylinositol 3'-kinase (PI3K), SHP-2 or phospholipase C-gamma. We found that PDGFRA-dependent cell survival was mainly mediated through activation of PI3K, and was negatively regulated by Src. Characterization of the downstream signaling events revealed that PI3K activates the protein kinase Akt, which in turn phosphorylates and thus inactivates proapoptotic Forkhead transcription factors. Src phosphorylates the ubiquitin-ligase c-Cbl, which is required for degradation of the activated receptor. Consequently, overexpression of c-Cbl prevented PDGFRA-mediated cell survival, whereas it did not affect this response, when Src was unable to associate with the receptor. This novel function of Src in antiapoptotic signaling introduces Src kinases as an interesting therapeutic target in apoptosis related diseases.

The Arg389Gly beta1-adrenoceptor gene polymorphism determines contractile response to catecholamines.

OBJECTIVES: Recently, the Arg389Gly beta1-adrenoceptor (beta1AR) gene polymorphism has been detected. The Arg variant exhibited increased responsiveness to agonist-induced stimulation in vitro. Functional studies in isolated human atrial muscle strips and in-vivo studies revealed contradictory results regarding the functional relevance of this polymorphism. We sought to characterize the functional consequences of the Arg389Gly beta1-AR polymorphism in 30 consecutive healthy male volunteers in vivo. METHODS: beta1-AR genotype was determined by PCR and restriction analysis, which was confirmed by DNA sequencing. We compared heart rate, blood pressure, and contractile response of the various genotype carriers with a modified dobutamine stress echocardiography protocol. RESULTS: Subjects homozygous for the Arg389 beta1AR showed a significantly higher increase in fractional shortening upon cumulative doses of dobutamine as compared to subjects carrying one or two copies of the Gly389 allele. A statistically significant difference was observed at a dobutamine dose of 10 microg/kg/min (46.5 +/- 1.3 vs. 41.8 +/- 1.0 %; P = 0.023) and was maximal at 40 microg/kg/min (61.9 +/- 1.4 vs. 52.8 +/- 1.6; P = 0.001). As a result, the systolic blood pressure response to dobutamine was significantly enhanced in individuals homozygous for the Arg389 allele, whereas the effect on heart rate did not differ between the two groups. Normalization for changing afterload conditions by calculating the pressure-dimension ratio revealed similar effects, indicating that the beta1AR-mediated effects are mainly a result of increased myocardial inotropy. CONCLUSION: These data indicate that the Arg389Gly beta1AR polymorphism is functionally relevant in vivo and determines contractile responsiveness to catecholamines in humans.