Direct noncompetitive inhibition of 5-HT₃ receptor-mediated responses by forskolin and steroids

5-HT₃ receptors cloned from NCB-20 cells were expressed in Xenopus oocytes, and the effects of forskolin and steroids on the function of the receptors were investigated using the two-electrode voltage-clamp technique. Forskolin, 17-β-estradiol, and progesterone inhibited the currents activated by 1 μM 5-HT in a reversible and concentration-dependent manner, with IC₅₀ values of 12, 33, and 89 μM, respectively. The inhibitory effects of forskolin and 17-β-estradiol were independent of the membrane potential. Forskolin and 17-β-estradiol significantly reduced the maximal amplitude of the 5-HT concentration-response curve (E_max) without significantly affecting the EC₅₀, indicating that these compounds act as noncompetitive inhibitors of the 5-HT₃ receptor. The cAMP analogue, 8-Br-cAMP (0.2mM), and the protein kinase A activator, Sp-cAMP (0.1 mM), did not affect the amplitude of 5-HT₃ receptor-mediated currents. The membrane-permeable protein kinase A inhibitor Rp-cAMP (0.1 mM) and the estrogen-receptor antagonist tamoxifen (1 μM) did not affect the inhibition of 5-HT-activated current. In addition, 5-HT₃ receptor-mediated currents were inhibited by both 1,9-dideoxy forskolin (30 μM), which does not activate adenylyl cyclase, and wForskolin (30 μM), a charged hydrophilic analogue of forskolin that is membrane impermeable. These results indicate that both forskolin and 17-β3-estradiol inhibit the function of the 5-HT₃ receptor in a noncompetitive manner and that this inhibition is independent of cAMP levels.