Regulation of the human Gi alpha-2 gene promotor activity in embryonic chicken cardiomyocytes.
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.
Eschenhagen
T
T
Abt. Allg. Pharmakologie Universitäts-Krankenhaus Eppendorf Univ. Hamburg.
Friedrichsen
M
M
Gsell
S
S
Hollmann
A
A
Mittmann
C
C
Schmitz
W
W
Scholz
H
H
Weil
J
J
Weinstein
L S
LS
D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002478:Cells, Cultured; D002642:Chick Embryo; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000235:genetics / Q000378:metabolism*; D006321:Heart / Q000187:drug effects / Q000196:embryology; D006801:Humans; D009154:Mutation; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D014162:Transfection; D015854:Up-Regulation / Q000235:genetics
D012333:RNA, Messenger; D005576:Colforsin; D003994:Bucladesine; D000242:Cyclic AMP; D015500:Chloramphenicol O-Acetyltransferase; D019206:GTP-Binding Protein alpha Subunits, Gi-Go