Overexpression of Gs alpha protein in the hearts of transgenic mice.

Gaudin, Christophe; Ishikawa, Yoshihiro; Wight, David C.; Mahdavi, Vijak; Nadal-Ginard, B; Wagner, Thomas E.; Vatner, Dorothy E.; Homcy, Charles J.

doi:10.1172/jci117843

Cited by 132 publications

(79 citation statements)

References 30 publications

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“…However, previous studies in the heart have shown that ␤AR overexpression does not yield a proportional increase in agonist-promoted stimulation of cAMP production (3,4). Similarly, overexpression of cardiac G␣s has limited effects on adrenergic responsiveness (5). In contrast, the present study indicates that overexpression of AC VI in cardiac myocytes is associated with a robust amplification of ␤AR-mediated signaling.…”

Section: Resultscontrasting

confidence: 75%

“…Variations in cell preparation, viral titer, and cAMP assays may contribute to the range in cAMP production that we found. Previous studies examining augmentation of transmembrane adrenergic signaling by altering ␤AR or Gs expression (3)(4)(5) or inhibiting G-protein coupled receptor kinase function (12) typically achieve no more than a 2-fold increase in cAMP production.…”

Section: Resultsmentioning

confidence: 99%

“…Thus, increased ␤AR number does not provide a proportional increase in transmembrane signaling through AC. Similarly, cardiac directed overexpression of G␣s in transgenic mice only modestly increased cardiac adrenergic responsiveness (5). In these studies a fixed amount of AC may have limited the degree to which increased ␤AR or Gs expression could provoke a meaningful physiological response.…”

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confidence: 95%

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Increased expression of adenylylcyclase type VI proportionately increases β-adrenergic receptor-stimulated production of cAMP in neonatal rat cardiac myocytes

Gao

Ping

Post

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

112

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Cellular content of cAMP generated by activation of adenylylcyclase (AC; EC 4.6.1.1) is a key determinant of functional responsiveness in the heart and other tissues. We have tested two hypotheses regarding the relationship between AC content and ␤-adrenergic receptor (␤AR)-mediated signal transduction in cardiac myocytes. First, that AC content limits adrenergic signal transduction, and, second, that increased AC, independent of (␤AR) number and G-protein content, yields a proportional increase in ␤AR-mediated transmembrane signaling. We used recombinant adenovirus to increase AC isoform VI (AC VI ) expression in neonatal cardiac myocytes. Cells that overexpressed AC VI responded to agonist stimulation with marked increases in cAMP production in proportion to protein expressed. In parallel experiments performed on cells transfected with lacZ (control) or AC VI , [ 3 H]forskolin binding, used to assess AC protein expression, was amplified 6-fold, while ␤AR-stimulated cAMP production from these cells was increased 7-fold. No changes in ␤AR number, or in the heterotrimeric GTP-binding proteins, G␣s or G␣i 2 , were observed. Previous studies indicate that increased cardiac expression of ␤AR or G␣s does not yield proportional increases in transmembrane adrenergic signaling. In contrast, the current data demonstrate that increased AC VI expression provides a proportional increase in ␤-adrenergic signal transduction. Our results show that the amount of AC sets a limit on transmembrane ␤-adrenergic signaling. We speculate that similar functional responses are possible in other cell types in which AC plays an important physiological role.

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Section: Resultscontrasting

confidence: 75%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 95%

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Increased expression of adenylylcyclase type VI proportionately increases β-adrenergic receptor-stimulated production of cAMP in neonatal rat cardiac myocytes

Gao

Ping

Post

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

112

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“…It has been shown that adenylyl cyclase is the critical component that limits maximal response to the ␤-adrenergic receptor (22). Thus, overexpression of the receptor (or G s ) in isolated cells (23) or transgenic animals (24) results in only modest enhancements in activation. We thus titrated the plasmid DNA used for our transfection (0.025-2.0 g of DNA/ ml) using equal concentrations for hIP1D4 and R212H (Fig.…”

Section: Fig 2 Competition Binding For Hip1d4pmt4 V25m and R212hmentioning

confidence: 99%

Impaired Receptor Binding and Activation Associated with a Human Prostacyclin Receptor Polymorphism

Stitham

Stojanović

Hwa

2002

Journal of Biological Chemistry

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The human prostacyclin receptor (hIP) is a seven transmembrane-spanning G-protein-coupled receptor that plays an important role in vascular homeostasis. Recent genetic analyses (SNP database, NCBI) have revealed the first two polymorphisms within the coding sequence, V25M and R212H. Here we present structurefunction characterizations of these polymorphisms at physiological pH (7.4) and at an acidic pH (6.8) that would be encountered during stress such as renal, respiratory, or heart failure. Through a series of competition binding and G-protein activation assays (measured by cAMP production), we determined that the V25M polymorph exhibited agonist binding and G-protein activation similar to wild-type receptor at normal pH (7.4). However, the R212H variant demonstrated a significant decrease in binding affinity at lower pH (R212H at pH 7.4, K i ‫؍‬ 2.2 ؎ 1.2 nM; pH 6.8 K i ‫؍‬ 45.6 ؎ 12.0 nM). The R212H polymorph also exhibited abnormal activation at both pH 7.4 and pH 6.8 (pH 7.4, R212H EC 50 ‫؍‬ 2.8 ؎ 0.5 nM versus wild-type hIP EC 50 ‫؍‬ 0.5 ؎ 0.1 nM; pH 6.8, R212H EC 50 ‫؍‬ 3.2 ؎ 1.6 nM versus wild-type hIP EC 50 ‫؍‬ 0.5 ؎ 0.2 nM). Polymorphisms of the human prostacyclin receptor potentially may be important predictors of disease progress during biological stressors such as acidosis in which urgent correction of bodily pH may be required to restore normal hemostasis and vasodilation. This study provides the mechanistic basis for further research into genetic risk factors and pharmacogenetics of cardiovascular disease associated with hIP.Similar to other prostanoids, prostacyclin is a derivative of the C-20 unsaturated fatty acid arachidonic acid (5,8,11,14-eicosatetraenoic acid), and its cellular action is conveyed through cell surface G-protein-coupled receptors that predominantly couple to the heterotrimeric G-protein G s stimulating the production of cAMP (1). The human prostacyclin receptor (hIP) 1 is expressed on platelets, where it mediates inhibition of platelet aggregation and on vascular smooth muscle cells, where it mediates vascular smooth muscle relaxation. Dysfunctional prostacyclin activity has been implicated in the development of a number of cardiovascular diseases including thrombosis, myocardial infarction, stroke, myocardial ischemia, atherosclerosis, and systemic and pulmonary hypertension (2). Accordingly, IP receptor knock-out mice exhibit increased thrombosis and reduced inflammatory and pain responses (3).Limited studies have begun to identify generalized regions within the IP and other prostanoid receptors that appear crucial for ligand-binding specificity and affinity. Studies using chimeric combinations of mouse prostaglandin D (mDP) and prostaglandin I (mIP) receptors have shown that protein segments within transmembrane domains VI and VII (TMVI and TMVII) are involved in distinct binding interactions with prostacyclin side chains. In addition, TMI (along with a portion of the first extracellular loop) confers broader binding functions, incorporating recognition and interaction w...

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“…19 We attempted to achieve cardiac specific gene expression by cloning the rat ␣-myosin heavy chain promoter (␣MyHC) in a recombinant adenovirus. To maximize cloning capacity, we have employed a short version of the ␣MyHC promoter (Ϫ613 to +32) that is sufficient for directing high levels of cardiac-specific gene expression in transgenic mice, 20,21 as well as after direct gene injection. 22 However, when a recombinant adenovirus carrying the ␣MyHC promoter driving the chloramphenicol report gene (CAT) was tested in vivo by direct injection, we found that CAT expression was not restricted to the heart, but was also detectable in several tissues (data not shown).…”

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confidence: 99%

Potential limitations of transcription terminators used as transgene insulators in adenoviral vectors

Buvoli

Bialik

et al. 2002

Gene Ther

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Several groups have modified adenoviral vector broad tropism and have achieved a targeted gene expression in somatic tissues using a variety of approaches including modification of viral structural proteins and inclusion of tissue-specific promoters. 1-9 However, the finding that the adenoviral background can cause either loss of tissue specificity [10][11][12] or down-regulation of promoters used to drive transgene expression 12 has limited the application of the last approach.Transcriptional interference mediated by several ciselements, mainly located on the left end of the adenovirus chromosome, appears to be accountable for this phenomenon (Figure 1a). The inverted terminal repeat (ITR) contains redundant binding sites for cellular factors and stimulates transcription from E1 and E4 genes. [13][14][15] The bipartite E1A enhancer trans-activates the E1A gene through the cellular protein EF-1A and controls the expression of the early genes after infection. 16,17 The promoter for the intermediate transcript pIX is situated just downstream of the canonical E1 cloning site for expression cassettes. Since some of these elements overlap with the viral origin of replication and the packaging signal, they cannot be deleted or inactivated. Thus other strategies based on the use of transcription modulators have been developed to achieve targeted gene delivery.

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Overexpression of Gs alpha protein in the hearts of transgenic mice.

Cited by 132 publications

References 30 publications

Increased expression of adenylylcyclase type VI proportionately increases β-adrenergic receptor-stimulated production of cAMP in neonatal rat cardiac myocytes

Increased expression of adenylylcyclase type VI proportionately increases β-adrenergic receptor-stimulated production of cAMP in neonatal rat cardiac myocytes

Impaired Receptor Binding and Activation Associated with a Human Prostacyclin Receptor Polymorphism

Potential limitations of transcription terminators used as transgene insulators in adenoviral vectors

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