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The hypocretins are weak agonists at recombinant human orexin-1 and orexin-2 receptors
D Smart1, J C Jerman, S J Brough
1Neuroscience Research, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK. Darren_2_Smart@sbphrd.com
Abstract:
The pharmacology of the orexin-like peptides, hypocretin-1 and hypocretin-2, was studied in Chinese hamster ovary (CHO) cells stably expressing orexin-1 (OX(1)) or orexin-2 (OX(2)) receptors by measuring intracellular calcium ([Ca(2+)](i)) using Fluo-3AM. Orexin-A and orexin-B increased [Ca(2+)](i) in CHO-OX(1) (pEC(50)=7. 99+/-0.05 and 7.00+/-0.10 respectively, n=8) and CHO-OX(2) (pEC(50)=8.30+/-0.05 and 8.21+/-0.07 respectively, n=5). However, hypocretin-1 and hypocretin-2 were markedly less potent, with pEC(50) values of 5.31+/-0.04 and 5.41+/-0.04 respectively in CHO-OX(2) cells (n=5). In CHO-OX(1) cells 10 microM hypocretin-1 only elicited a 37.5+/-3.4% response whilst 10 microM hypocretin-2 elicited a 18.0+/-2.1% response (n=8). Desensitisation of OX(1) or OX(2) with orexin-A (100 nM) abolished the response to orexin-A (10 nM) and the hypocretins (10 microM), but not to UTP (3 microM). In conclusion, the hypocretins are only weak agonists at the orexin receptors.
Insights
Hypocretins are weak agonists at orexin receptors. Orexin-A and orexin-B effectively activate orexin-1 (OX(1)) and orexin-2 (OX(2)) receptors, while hypocretins show minimal potency.
Area of Science:
- Pharmacology
- Neuroscience
- Cell Biology
Background:
- Orexin-like peptides, hypocretin-1 and hypocretin-2, are involved in regulating various physiological functions.
- Orexin receptors (OX(1) and OX(2)) mediate the actions of orexin peptides.
Purpose of the Study:
- To investigate the pharmacological profiles of hypocretin-1 and hypocretin-2 at OX(1) and OX(2) receptors.
- To compare the potency of hypocretins with orexin-A and orexin-B.
Main Methods:
- Utilized Chinese hamster ovary (CHO) cells stably expressing OX(1) or OX(2) receptors.
- Measured intracellular calcium ([Ca(2+)](i)) increase using Fluo-3AM as an indicator of receptor activation.
- Performed receptor desensitization experiments with orexin-A.
Main Results:
- Orexin-A and orexin-B demonstrated significant potency at both OX(1) and OX(2) receptors (pEC(50) values ranging from 7.00 to 8.30).
- Hypocretin-1 and hypocretin-2 exhibited markedly lower potency, particularly at OX(2) receptors (pEC(50) values around 5.3-5.4).
- Hypocretins elicited only partial responses in CHO-OX(1) cells, even at high concentrations, and receptor desensitization experiments confirmed their weak agonistic activity.
Conclusions:
- Hypocretins act as weak agonists at both orexin-1 and orexin-2 receptors.
- Orexin-A and orexin-B are potent agonists, highlighting distinct pharmacological profiles compared to hypocretins.