Regulation of the human Gi alpha-2 gene promotor activity in embryonic chicken cardiomyocytes.

Abstract

Increased expression of the inhibitory G protein Gi alpha-2 is assumed to contribute to desensitization of adenylyl cyclase in human heart failure. The mechanisms of upregulation involve increases in myocardial Gi alpha-2 protein, mRNA and gene transcriptional activity. To elucidate these mechanisms in more detail, the 5' flanking region of the human Gi alpha-2 gene (-1214/+115 bp) was cloned upstream of the bacterial chloramphenicol acetyltransferase (CAT) gene and transfected in embryonic chick cardiomyocytes. CAT activity was measured 48 h after transfection. Unstimulated activity of the -1214/+115 bp construct was about 10-fold higher than activity of the basal CAT-construct (pGEMCAT). 5' deletion from -1214/+115 to -85/+115 bp upstream of the transcriptional start site increased, further stepwise deletions to 46/+115 gradually decreased promotor activity. Deletion from -46/+115 to -33/+115 bp completely abolished promotor activity. Stimulation of cardiomyocytes that had been transfected with the -1214/+115 CAT-construct with isoprenaline (10 microM), forskolin (10 microM), forskolin (10 microM) plus IBMX (10 microM) or dibutyryl-cAMP (1 mM) for 24 h induced an increase in CAT activity to 139 +/- 12% (n = 9), 211 +/- 18% (n = 12), 256 +/- 20% (n = 5) and 198 +/- 28% (n = 7) of unstimulated values, respectively. We conclude: 1) In chicken cardiomyocytes a sequence element of 52 bp between -85 and -33 bp is necessary to provide basal Gi alpha-2 promotor activity. 2) Elevation of cAMP has a stimulatory effect on the human Gi alpha-2 promotor, thereby offering a mechanism for beta-adrenoceptor-mediated increases in Gi alpha-2 in the heart.

• Authors: T Eschenhagen, M Friedrichsen, S Gsell, A Hollmann, C Mittmann, W Schmitz, H Scholz, J Weil, L S Weinstein
• Journal: Basic research in cardiology (Basic Res Cardiol) Vol. 91 Suppl 2 Pg. 41-6 ( 1996) ISSN: 0300-8428 [Print] Germany
• PMID: 8957543 (Publication Type: Journal Article)

Chemical References

• RNA, Messenger
• Colforsin
• Bucladesine
• Cyclic AMP
• Chloramphenicol O-Acetyltransferase
• GTP-Binding Protein alpha Subunits, Gi-Go

Topics

• Animals
• Bucladesine (pharmacology)
• Cells, Cultured
• Chick Embryo
• Chloramphenicol O-Acetyltransferase (metabolism)
• Colforsin (pharmacology)
• Cyclic AMP (metabolism)
• GTP-Binding Protein alpha Subunits, Gi-Go (genetics, metabolism)
• Heart (drug effects, embryology)
• Humans
• Mutation
• Myocardium (cytology, metabolism)
• Plasmids (genetics)
• Promoter Regions, Genetic
• RNA, Messenger (genetics)
• Transfection
• Up-Regulation (genetics)