ENHANCED BRADYKININ-STIMULATED PHOSPHOLIPASE-C ACTIVITY IN MURINE EMBRYONIC STEM-CELLS LACKING THE G-PROTEIN ALPHA(Q)-SUBUNIT

The gene coding for the G-protein alpha(q) subunit was interrupted by homologous recombination in murine embryonic stem cells (alpha(q)-null ES cells) as detected by Southern analysis and reverse-transcriptase PCR. The bradykinin (BK) B2 receptor was stably transfected into wild- type (WT) alpha(i-2)-null and alpha(q)-null ES cells. The B2 receptor bound BK with high affinity and mobilized Ca2+. BK also activated phos pholipase C (PLC), as determined by total inositol phosphate (IP) accu mulation in a Bordetella pertussis toxin- and genistein-insensitive ma nner. In WT and alpha(i-2)-null ES cells, BK increased IP levels appro x. 4-fold above baseline. Most interestingly, in alpha(q)-null ES cell s, BK increased IP accumulation approx. 9-fold above baseline. Re-expr ession of alpha(q) in alpha(q)-nulI ES cells resulted in normalization of the BK-stimulated IP accumulation (4-fold above baseline). These r esults suggest that the B2 receptor activates PLC through more than on e member of the G(q) family. Additionally, the absence of alpha(q) alt ers the kinetics of IP generation, which may reflect intrinsic charact eristics of individual members of the G(q) family or a decreased susce ptibility to heterologous regulation in the alpha(q)-nulI ES cells, th us allowing for a more sustained generation of IP.