Premium
Coupling of an endogenous 5‐HT 1B ‐like receptor to increases in intracellular calcium through a pertussis toxin‐sensitive mechanism in CHO‐K1 cells
Author(s) -
Dickenson John M.,
Hill Stephen J.
Publication year - 1995
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/j.1476-5381.1995.tb15941.x
Subject(s) - pertussis toxin , chinese hamster ovary cell , adenylyl cyclase , forskolin , medicine , endocrinology , receptor , intracellular , biology , extracellular , calcium in biology , ec50 , chemistry , g protein , biochemistry , in vitro
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 A 1 receptor when expressed in CHO‐K1 cells (CHO‐A1) has been shown to mobilize intracellular Ca 2+ through a PTX‐sensitive mechanism. Therefore the aim of this investigation was to determine whether the endogenous 5‐HT 1B ‐like receptor was able to stimulate increases in intracellular free [Ca 2+ ] ([Ca 2+ ] 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 [ 3 H]‐cyclic AMP production in CHO‐A1 cells (p[EC 50 ] = 7.73±0.13). 5‐HT (1 μ m ) inhibited 47±5% of the [ 3 H]‐cyclic AMP accumulation induced by 3 μ m forskolin. Forskolin stimulated [ 3 H]‐cyclic AMP accumulation was also inhibited by the 5‐HT 1 receptor agonists (p[EC 50 ] 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 [Ca 2+ ] i in CHO‐A1 cells (p[EC 50 ] = 8.07 ±0.05). In the presence of 2mM extracellular Ca 2+ , 5‐HT (1 μ m ) increased [Ca 2+ ] i from 174±17nM to 376 ±22 nM. The 5‐HT 1 receptor agonists (p[EC 50 ] 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 [Ca 2+ ] i . Similar maximal increases in [Ca 2+ ] i were obtained with each agonist. The selective 5‐HT 1A receptor agonist, 8‐OH‐DPAT (10 μ m ) did not stimulate increases in [Ca 2+ ] i . 5‐HT (100 μ m ) and 5‐CT (10 μ m ) did not stimulate a measurable increase in [ 3 H]‐inositol phosphate accumulation in CHO‐A1 cells. 4 5‐HT (1 μ m )‐mediated increases in [Ca 2+ ] i were insensitive to the 5‐HT receptor antagonist, ritanserin (5‐HT 2 ; 100 nM), ketanserin (5‐HT 2 ; 100 nM), LY‐278,584 (5‐HT 3 ; 1 μ m ) and WAY 100635 (5‐HT 1A ; 1 μ m ). The response to 5‐HT (100 nM) was antagonized by the non‐selective 5‐HT 1 antagonist, methiothepin (pK b = 8.90±0.09) and the 5‐HT 1D antagonist GR 127935 (pK b = 10.44±0.06). 5 Pretreatment with PTX (200 ng ml −1 for 4 h) completely attenuated the Ca 2+ response to 100 μ m 5‐HT. 6 In untransfected CHO‐K1 cells, 5‐HT (1 μ m ), RU 24969 (1 μ m ), and 5‐CT (1 μ m ) elicited increases in [Ca 2+ ] i similar to those observed in CHO‐A1 cells. 7 These data demonstrate that in CHO‐K1 cells the endogenously expressed 5‐HT 1B ‐like receptor couples to the phospholipase C/Ca 2+ signalling pathway through a PTX‐sensitive pathway, suggesting the involvement of G i /G o protein(s).