Coupling of an endogenous 5‐HT1B‐like receptor to increases in intracellular calcium through a pertussis toxin‐sensitive mechanism in CHO‐K1 cells

Abstract

Chinese hamster ovary cells (CHO‐K1) express an endogenous 5‐hydroxytryptamine (5‐HT)1B‐like receptor that is negatively coupled to adenylyl cyclase through a pertussis toxin (PTX)‐sensitive mechanism. Furthermore, the human adenosine A1 receptor when expressed in CHO‐K1 cells (CHO‐A1) has been shown to mobilize intracellular Ca2+ through a PTX‐sensitive mechanism. Therefore the aim of this investigation was to determine whether the endogenous 5‐HT1B‐like receptor was able to stimulate increases in intracellular free [Ca2+] ([Ca2+]i) in CHO‐A1 cells. In agreement with previous studies using CHO cells, 5‐hydroxytryptamine (5‐HT) elicited a concentration‐dependent inhibition of forskolin‐stimulated [3H]‐cyclic AMP production in CHO‐A1 cells (p[EC50] = 7.73±0.13). 5‐HT (1 μm) inhibited 47±5% of the [3H]‐cyclic AMP accumulation induced by 3 μm forskolin. Forskolin stimulated [3H]‐cyclic AMP accumulation was also inhibited by the 5‐HT1 receptor agonists (p[EC50] values) 5‐carboxyamidotryptamine (5‐CT; 8.07±0.08), RU 24969 (8.12 ±0.33) and sumatriptan (5.80 ±0.31). 5‐HT elicited a concentration‐dependent increase in [Ca2+]i in CHO‐A1 cells (p[EC50] = 8.07 ±0.05). In the presence of 2mM extracellular Ca2+, 5‐HT (1 μm) increased [Ca2+]i from 174±17nM to 376 ±22 nM. The 5‐HT1 receptor agonists (p[EC50] values), 5‐carboxyamidotryptamine (5‐CT; 7.9 ±0.02), RU 24969 (8.1 ±0.07) and sumatriptan (5.9 ±0.11) all elicited concentration‐dependent increases in [Ca2+]i. Similar maximal increases in [Ca2+]i were obtained with each agonist. The selective 5‐HT1A receptor agonist, 8‐OH‐DPAT (10 μm) did not stimulate increases in [Ca2+]i. 5‐HT (100 μm) and 5‐CT (10 μm) did not stimulate a measurable increase in [3H]‐inositol phosphate accumulation in CHO‐A1 cells. 5‐HT (1 μm)‐mediated increases in [Ca2+]i were insensitive to the 5‐HT receptor antagonist, ritanserin (5‐HT2; 100 nM), ketanserin (5‐HT2; 100 nM), LY‐278,584 (5‐HT3; 1 μm) and WAY 100635 (5‐HT1A; 1 μm). The response to 5‐HT (100 nM) was antagonized by the non‐selective 5‐HT1 antagonist, methiothepin (pKb = 8.90±0.09) and the 5‐HT1D antagonist GR 127935 (pKb= 10.44±0.06). Pretreatment with PTX (200 ng ml−1 for 4 h) completely attenuated the Ca2+ response to 100 μm 5‐HT. In untransfected CHO‐K1 cells, 5‐HT (1 μm), RU 24969 (1 μm), and 5‐CT (1 μm) elicited increases in [Ca2+]i similar to those observed in CHO‐A1 cells. These data demonstrate that in CHO‐K1 cells the endogenously expressed 5‐HT1B‐like receptor couples to the phospholipase C/Ca2+ signalling pathway through a PTX‐sensitive pathway, suggesting the involvement of Gi/Go protein(s). 1995 British Pharmacological Society