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

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Abstract
1 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. 2 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). 3 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. 4 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). 5 Pretreatment with PTX (200 ng ml−1 for 4 h) completely attenuated the Ca2+ response to 100 μm 5-HT. 6 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. 7 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).