Strains within the genus Salinospora have been shown to produce complex natural products having antibiotic and antiproliferative activities. The biochemical basis for the cytotoxic effects of salinosporamide A has been linked to its ability to inhibit the proteasome. Synthetically accessible salinosporamide A (ML858) was used to determine its biochemical and biological activities and to compare its effects with those of bortezomib. ML858 and bortezomib show time-and concentration-dependent inhibition of the proteasome in vitro. However, unlike bortezomib, which is a reversible inhibitor, ML858 covalently binds to the proteasome, resulting in the irreversible inhibition of 20S proteasome activity. ML858 was equipotent to bortezomib in cell-based reporter stabilization assays, but due to intramolecular instability is less potent in long-term assays. ML858 failed to maintain levels of proteasome inhibition necessary to achieve efficacy in tumor models responsive to bortezomib. Our results show that ML858 and bortezomib exhibit different kinetic and pharmacologic profiles and suggest that additional characterization of ML858 is warranted before its therapeutic potential can be fully appreciated. [Mol Cancer Ther 2006;5(12):3052-61]
Whereas many cardiac symptoms of thyrotoxicosis resemble those of the hyperadrenergic state, circulating catecholamines are reduced or normal in this condition. To test the hypothesis that the thyrotoxic heart is hypersensitive to catechol-amines, we studied beta-adrenergic signaling in a transgenic (TG) mouse in which the human type 2 iodothyronine deiodinase (D2) gene is expressed in myocardium. Because D2 converts T4 to T3, the active form of thyroid hormone, the D2 TG mouse exhibits mild, chronic thyrotoxicosis that is limited to the myocardium. In the current study, we determined that cAMP accumulation in response to either norepinephrine or forskolin treatment was increased in isolated ventricular myocardiocytes and membrane-enriched fractions prepared from these D2 TG hearts as compared with wild type. This increase in adenylyl cyclase (AC) Vmax could not be explained by changes in AC isoform expression or changes in the long or short forms of stimulatory G-protein Gsalpha, which were approximately 10% decreased in D2 TG membranes. However, Western analysis and ADP-ribosylation studies suggest that the increase in AC Vmax is mediated by a decrease in the expression of inhibitory G proteins (Gialpha-3 and/or Goalpha). These data suggest that cardiac thyrotoxicosis leads to increased beta-adrenergic responsiveness of cardiomyocytes via alterations in the regulatory G-protein elements of the AC membrane complex.
In contrast to the other heterotrimeric GTP-binding proteins (G proteins) G s and Gi, the functional role of Go is still poorly defined. To investigate the role of G␣o in the heart, we generated transgenic mice with cardiac-specific expression of a constitutively active form of G␣ o1 ء (G␣ o ,)ء the predominant G␣ o isoform in the heart. G␣ o expression was increased 3-to 15-fold in mice from 5 independent lines, all of which had a normal life span and no gross cardiac morphological abnormalities. We demonstrate enhanced contractile function in G␣ oء transgenic mice in vivo, along with increased L-type Ca 2ϩ channel current density, calcium transients, and cell shortening in ventricular G␣ o-ءexpressing myocytes compared with wild-type controls. These changes were evident at baseline and maintained after isoproterenol stimulation. Expression levels of all major Ca 2ϩ handling proteins were largely unchanged, except for a modest reduction in Na ϩ /Ca 2ϩ exchanger in transgenic ventricles. In contrast, phosphorylation of the ryanodine receptor and phospholamban at known PKA sites was increased 1.6-and 1.9-fold, respectively, in G␣ oء ventricles. Density and affinity of -adrenoceptors, cAMP levels, and PKA activity were comparable in G␣ oء and wild-type myocytes, but protein phosphatase 1 activity was reduced upon G␣ oء expression, particularly in the vicinity of the ryanodine receptor. We conclude that G␣ oء exerts a positive effect on Ca 2ϩ cycling and contractile function. Alterations in protein phosphatase 1 activity rather than PKA-mediated phosphorylation might be involved in hyperphosphorylation of key Ca 2ϩ handling proteins in hearts with constitutive G␣ o activation. G proteins; signal transduction; calcium; contraction; transgenic mice CARDIAC CONTRACTILE FUNCTION is determined by the intrinsic contractile properties of the heart and is subject to neurohumoral regulation. The main receptors involved in regulating contraction are prototypical G protein-coupled receptors (42). They activate heterotrimeric G proteins that are comprised of ␣-, -, and ␥-subunits (40). G proteins are classified according to their ␣-subunits, because they primarily determine downstream signaling specificity. Several different G proteins are expressed in the heart (53). Members of the G s and G i/o subfamilies play a key role in transmitting extracellular signals that regulate myocyte cell shortening (42): 1) activation of G s protein-coupled receptors (such as -adrenergic receptors) leads to increased adenylyate cyclase activity and cAMP and PKA activation. PKA then phosphorylates several Ca 2ϩ handling and contractile proteins, resulting in increased contraction and relaxation. 2) Activation of cardiac receptors that are coupled to members of the pertussis toxin-sensitive G i/o subfamily (such as A 1 adenosine and M 2 muscarinic receptors) negatively regulate contractile function in mammalian ventricles in the presence of elevated cAMP ("accentuated antagonism"). While G␣ i has the capacity to directly inhib...
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