CHARACTERIZATION OF A 5-HT1B RECEPTOR ON CHO CELLS - FUNCTIONAL-RESPONSES IN THE ABSENCE OF RADIOLIGAND BINDING

1 Chinese hamster ovary (CHO) cells have been reported to be devoid of 5-HT receptors and have frequently been used as hosts for the express ion of cloned 5-HT receptors. Unexpectedly, 5-HT was found to induce p rofound inhibition of forskolin-stimulated cyclic AMP production in th ese cells and the aim of this study was to classify the 5-HT receptor involved. 2 In CHO(dhfr-) cells 5-HT was a potent agonist and caused 8 0-100% inhibition of forskolin stimulated cyclic AMP production. A stu dy using several 5-HT, receptor agonists revealed the following potenc ies (p[A(50)]): RU24969 (9.09 +/- 0.17) > 5-carboxamidotryptamine (8.8 6 +/- 0.20) > 5-HT (8.07 +/- 0.05) > CP-93,129 (7.74 +/- 0.10) > sumat riptan (5.93 +/- 0.04). All five agonists achieved a similar maximum e ffect. Irreversible receptor alkylation studies yielded a pK(A) estima te of 7.04 +/- 0.34 for 5-HT. 3 The 5-HT1A/1B antagonist, (+/-)-cyanop indolol (4-100 nM), caused parallel rightward shifts of the 5-HT conce ntration-effect curve with no change in asymptote. Schild analysis yie lded a pK(B) estimate of 8.69 +/- 0.09 (Schild slope 1.13 +/- 0.10). ( +/-)-Cyanopindolol actually behaved as a partial agonist with an intri nsic activity of 0.2-0.5 and a p[A(50)] of 8.55. 4 5-HT (0.01-10 mu M) also elicited a concentration-dependent increase in intracellular [Ca 2+] in CHO(dhfr-) cells thus demonstrating that dual coupling is not a phenomenon restricted to systems in which there is overexpression of transfected receptors. 5 This agonist and antagonist profile is consis tent with the presence of a 5-HT1B receptor. 8-OH-DPAT (1 mu M) and re nzapride (3 mu M) were without effect on forskolin-stimulated cyclic A MP production and ketanserin (0.3 mu M) did not antagonize the inhibit ion produced by 5-HT, thus excluding the involvement of 5-HT1A, 5-HT4, and 5-HT2 receptors. 6 The possibility that expression of a 5-HT1B re ceptor was associated with the dhfr- mutation was excluded since RU249 69, 5-HT and CP-93,129 were also potent agonists in unmutated, CHO-K1 cells: P[A(50)] 9.03 +/- 0.03, 8.34 +/- 0.05, 7.69 +/- 0.07 respective ly, and (+/-)-cyanopindolol (0.1 mu M) shifted the 5-HT curve to the r ight and yielded a pA(2) estimate of 8.70 +/- 0.06. 7 Little or no spe cific binding of [H-3]-5-HT (0.1-200 nM) or of the high affinity ligan d [I-125]-iodocyanopindolol (0.01-3 nM) to CHO(dhfr-) cell membranes c ould be detected. 5-HT also failed to elicit any increase in the bindi ng of [S-35]-GTP gamma S to CHO membranes. 8 In conclusion, cultured C HO cells express 5-HT1B receptors which are negatively coupled to aden ylyl cyclase and positively coupled to increases in intracellular calc ium. The absence of radioligand binding was unexpected in view of the high potency of 5-HT and the partial agonist activity of the normally 'silent' competitive antagonist, (+/-)-cyanopindolol. This implies ver y efficient receptor-effector coupling of a low density of 5-HT1B rece ptors. Clearly, the absence of detectable radioligand binding cannot b e assumed to mean the absence of receptors capable of eliciting a sign ificant functional response.