A ROLE FOR CAMP IN ANGIOTENSIN-II MEDIATED INHIBITION OF CELL-GROWTH IN AT(1A) RECEPTOR-TRANSFECTED CHO-K1 CELLS

G-protein coupled Angiotensin II receptors (AT(1A)), mediate cellular responses through multiple signal transduction pathways. InAT(1A) rece ptor-transfected CHO-K1 cells (T3CHO/AT(1A)), angiotensin II (AII) sti mulated a dose-dependent (EC(50) = 3.3 nM) increase in cAMP accumulati on, which was inhibited by the selective AT(1), nonpeptide receptor an tagonist EXP3174. Activation of protein kinase C, or increasing intrac ellular Ca2+ with ATP, the calcium ionophore A23187 or ionomycin faile d to stimulate cAMP accumulation. Thus, AII-induced cAMP accumulation was not secondary to activation of a protein kinase C- or Ca2+/calmodu lin-dependent pathway. Since cAMP has an established role in cellular growth responses, we investigated the effect of the AII-mediated incre ase in cAMP on cell number and [H-3]thymidine incorporation in T3CHOA/ AT(1A) cells. AII (1 mu M) significantly inhibited cell number (51% at 96 h) and [H-3]thymidine incorporation (68% at 24 h) compared to vehi cle controls. These effects were blocked by EXP3174, confirming that t hese responses were mediated through the AT(1) receptor. Forskolin (10 mu M) and the cAMP analog dibutyryl-cAMP (1 mM) also inhibited [H-3]t hymidine incorporation by 55 and 25% respectively. We extended our inv estigation on the effect of AII-stimulated increases in cAMP, to deter mine the role for established growth related signaling events, i.e., m itogen-activated protein kinase activity and tyrosine phosphorylation of cellular proteins. AII-stimulated mitogen-activated protein kinase activity and phosphorylation of the 42 and 44 kD forms. These events w ere unaffected by forskolin stimulated increases in cAMP, thus the AII -stimulated mitogen-activated protein kinase activity was independent of cAMP in these cells. AII also stimulated tyrosine phosphorylation o f a number of cellular proteins in T3CHO/AT(1A) cells, in particular a 127 kD protein. The phosphorylation of the 127 kD protein was transie nt, reaching a maximum at 1 min, and returning to basal levels within 10 min. The dephosphorylation of this protein was blocked by a selecti ve inhibitor of cAMP dependent protein kinase A, H89-dihydrochloride a nd preexposure to forskolin prevented the AII-induced transient tyrosi ne phosphorylation of the 127 kD protein. These data suggest that cAMP , and therefore protein kinase A can contribute to AII-mediated growth inhibition by stimulating the dephosphorylation of substrates that ar e tyrosine phosphorylated in response to AII.