5-HYDROXYTRYPTAMINE TYPE 2A RECEPTORS REGULATE CYCLIC-AMP ACCUMULATION IN A NEURONAL CELL-LINE BY PROTEIN-KINASE C-DEPENDENT AND CALCIUM CALMODULIN-DEPENDENT MECHANISMS/

The effects of 5-hydroxytryptamine (5-HT)(2A) receptor activation on c AMP formation were studied in a cell line derived from embryonic rat c ortex (A1A1). 5-HT (EC(50) = 0.87 mu M) amplified the amount of cAMP f ormed in response to 5'-N-ethylcarboxamidoadenosine (an adenosine A(2) receptor agonist), cholera toxin, and forskolin after 15 min of coinc ubation in the presence of the phosphodiesterase inhibitor rolipram. T his effect of 5-HT was blocked by 10 nM ketanserin as well as by 10 nM spiperone, indicating a response mediated by the 5-HT2A receptor subt ype. Similarly, cAMP accumulation was enhanced by coincubation with th e protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PM A) and the calcium ionophore A23187. After exposure to PMA for 24 hr ( PKC-depleted cells), 5-HT and A23187 still enhanced cAMP formed in res ponse to forskolin and 5'-N-ethylcarboxamidoadenosine, whereas the amp lifying effects of PMA were abolished. Analysis by Western blots and P KC activity measurements revealed that, of three PKC isoforms detected in A1A1 cells (alpha, delta, and epsilon), only the calcium-independe nt isoform PKC-epsilon remained in membrane fractions after long term PMA treatment. In PKC-depleted cells, 5-HT-mediated amplification was greatly reduced after treatment with the calcium chelator ,2-bis(o-ami nophenoxy)ethane-N,N,N',N'-tetraacetic acid (acetoxymethyl)-ester or t he calmodulin antagonists calmidazolium and N(6-aminohexyl)-5-chloro-1 -napthalenesulfonamide hydrochloride. In addition, 5-HT-mediated ampli fication of cAMP accumulation was reduced by the PKC inhibitor stauros porine in normal cells but was unaffected in PKC-depleted cells. In co nclusion, these data suggest that 5-HT2A receptor activation can ampli fy cAMP formation in A1A1 cells by two distinct pathways coupled to th e hydrolysis of inositol phosphates, i.e., PKC and calcium/calmodulin.