Abstract
In cell systems where ligand-independent receptor activity is optimized (such as when receptors are overexpressed or mutated), acute treatment with inverse agonists reduces basal effector activity whereas prolonged exposure leads to sensitization of receptor systems and receptor up-regulation. Few studies, however, have reported effects of inverse agonists in systems where nonmutated receptors are expressed at relatively low density. Here, we investigated the effects of inverse agonists at human serotonin (5-HT)2C receptors expressed stably in Chinese hamster ovary cells (≈250 fmol/mg protein). In these cells, there is no receptor reserve for 5-HT and 5-HT2C inverse agonists did not reduce basal inositol phosphate (IP) accumulation nor arachidonic acid (AA) release but behaved as simple competitive antagonists, suggesting that these receptors are not overexpressed. Prolonged treatment (24 h) with inverse agonists enhanced selectively 5-HT2C-mediated IP accumulation but not AA release. The enhancing effect occurred within 4 h of treatment, reversed within 3 to 4 h (after 24-h treatment), and could be blocked with neutral antagonists or weak positive agonists. The enhanced responsiveness was not due to receptor up-regulation but may involve changes in the expression of the G protein, Gαq/11 and possibly Gα12 and Gα13. Interestingly, 24-h exposure to inverse agonists acting at 5-HT2C receptors also selectively enhanced IP accumulation, but not AA release, elicited by activation of endogenous purinergic receptors. These data suggest that actions of inverse agonists may be mediated through effects on receptor systems that are not direct targets for these drugs.
Footnotes
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Send reprint requests to: Kelly A. Berg, Department of Pharmacology, University of Texas Health Science Center, 7703 Floyd Curl Dr., San Antonio, TX 78284-7764. E-mail: berg{at}uthscsa.edu
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↵1 It should be noted however, that Gether et al. (1997) reported that agonists, inverse agonists, and neutral antagonists could up-regulate expression of constitutively active mutants of the β2-adrenergic receptor in Sf9 cells when cells were incubated with ligand during the 48-h infection period. Similar results were reported by Samama et al. (1997) for β2 constitutively active mutants expressed in hearts of transgenic mice. These results were interpreted as due to biochemical stabilization of an inherently unstable, constitutively active receptor. Thus, at least in this treatment paradigm with mutant receptors, up-regulation of receptor density may occur through mechanisms other than by reduction in the activity of effector mechanisms involved in down-regulation.
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This work was supported by United States Public Health Service Grants DA 09094 (K.A.B., S.M.) and HD 26437 (W.P.C.). Portions of this work have been presented at the first International Union of Pharmacology Conference on Receptor Mechanisms: Principles of Agonism, Merano, Italy (1998), the fourth International Union of Pharmacology Satellite Meeting on Serotonin, Rotterdam, the Netherlands (1998) and at the annual meetings of the Society for Neuroscience (1997, 1998).
- Abbreviations:
- 5-HT
- serotonin
- 5-MXG
- 5-methoxygramine
- AA
- arachidonic acid
- BOL
- bromo-lysergic acid diethylamide
- CHO
- Chinese hamster ovary
- DMSO
- dimethyl sulfoxide
- DOI
- (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane
- HBSS
- Hanks’ balanced salt solution
- IP
- inositol phosphate
- PKC
- protein kinase C
- PLA2
- phospholipase A2
- PLC
- phospholipase C
- 5-HT2C
- serotonin type 2C
- BSA
- bovine serum albumin
- PVDF
- polyvinylidene fluoride
- PBS-T
- PBS containing 0.01% Tween-20
- Received April 1, 1998.
- Accepted January 27, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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