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 f ailure. The mechanisms of upregulation involve increases in myocardial Gi alpha-2 protein, mRNA and gene transcriptional activity. To elucid ate these mechanisms in more detail, the 5' flanking region of the hum an Gi alpha-2 gene (-1214/ + 115 bp) was cloned upstream of the bacter ial chloramphenicol acetyltransferase (CAT) gene and transfected in em bryonic chick cardiomyocytes, CAT activity was measured 48 h after tra nsfection. 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 tran scriptional 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-con struct with isoprenaline (10 mu M), forskolin (10 mu M), forskolin (10 mu M) plus IBMX (10 mu M) or dibutyryl-cAMP (1 mM) for 24 h induced a n increase in CAT activity to 139 +/- 12 % (n = 9), 211 +/- 18 % (n = 12), 256 +/- 20 % (n = 5) and 198 +/- 28 % (n = 7) of unstimulated val ues, respectively. We conclude: 1) In chicken cardiomyocytes a sequenc e clement of 52 bp between -85 and -33 bp is necessary to provide basa l 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.