WAY‐100635 antagonist‐induced plasticity of 5‐HT1A receptors: regulatory differences between a stable cell line and an in vivo native system

We present evidence that the 5-hydroxytryptamine1A (5-HT1A) receptor antagonist, N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]-ethyl}-N-(2-pyridinyl)cyclohexanecarboxamide (WAY-100635), can induce receptor internalization in a human (h)5-HT1A receptor Chinese hamster ovary (CHO-K1) cell system. Exposure of h5-HT1A CHO cells to WAY-100635 decreased the cell-surface h5-HT1A receptor density in a way that was both time (24–72 h) and concentration (1–100 nm) dependent.[3H]WAY-100635 and [3H]8-hydroxy-dipropylaminotetralin ([3H]8-OH-DPAT) saturation analyses demonstrated a significant reduction (50–60%) in total h5-HT1A receptor number in the WAY-100635-treated (100 nm; 72 h) compared with control cells. In WAY-100635-treated cells, the 8-OH-DPAT-mediated inhibition of forskolin (FSK)-stimulated cAMP accumulation was right-shifted and the maximal inhibitory response of 8-OH-DPAT was impaired compared with control cells. Similar results were obtained for 8-OH-DPAT-mediated Ca2+ mobilization after WAY-100635 treatment. h5-HT1A receptors labeled with [3H]WAY-100635, as well as [3H]4-(2′-Methoxy)-phenyl-1-[2′-(N-2′′-pyridinyl)-p-fluorobenzamido]ethyl-piperazine (MPPF), exhibited a time-dependent rate of cellular internalization that was blocked by endocytotic suppressors and was pertussis-toxin insensitive. In contrast, quantitative autoradiographic studies demonstrated that chronic treatment of rats with WAY-100635 for two weeks produced a region-specific increase in the 5-HT1A receptor density. In conclusion, prolonged exposure of an h5-HT1A cell-based system to the 5-HT1A antagonist, WAY-100635, induced a paradoxical internalization of cell surface receptor resulting in depressed functional activity. This suggests that an antagonist can influence 5-HT1A receptor recycling in vitro differently to in vivo regulatory conditions.