h5‐HT_1B receptor‐mediated constitutive Gα_i3‐protein activation in stably transfected Chinese hamster ovary cells: an antibody capture assay reveals protean efficacy of 5‐HT

Q2 · MEDICINE

Article

Author: Marini, Laetitia ; Millan, Mark J. ; Touzard, Manuelle ; Newman-Tancredi, Adrian ; Cussac, Didier

Serotonin 5‐HT1B receptors couple to G‐proteins of the Gi/o family. However, their activation of specific G‐protein subtypes is poorly characterised. Using an innovative antibody capture/guanosine‐5′‐0‐(3‐[35S]thio)‐triphosphate ([35S]GTPγS) binding strategy, we characterised Gαi3 subunit activation by h5‐HT1B receptors stably expressed in Chinese hamster ovary (CHO) cells.

The agonists, 5‐HT, alniditan and BMS181,101, stimulated Gαi3, whereas methiothepin and SB224,289 behaved as inverse agonists. The selective 5‐HT1B receptor ligand, S18127, modestly stimulated Gαi3 and reversed the actions of both 5‐HT and methiothepin. S18127 (1 μM) also produced parallel, dextral shifts of the 5‐HT and methiothepin isotherms.

Isotopic dilution experiments ([35S]GTPγS versus GTPγS) revealed high‐affinity [35S]GTPγS binding to Gαi3 subunits in the absence of receptor ligands indicating constitutive activity. High‐affinity [35S]GTPγS binding was increased 2.8‐fold by 5‐HT with an increase in the affinity of GTPγS for Gαi3 subunits. In contrast, methiothepin halved the number of high‐affinity binding sites and decreased their affinity.

h5‐HT1B receptor‐mediated Gαi3 subunit activation was dependent on the concentration of NaCl. At 300 mM, 5‐HT stimulated [35S]GTPγS binding, basal Gαi3 activation was low and methiothepin was inactive. In contrast, at 10 mM NaCl, basal activity was enhanced and the inverse agonist activity of methiothepin was accentuated. Under these conditions, 5‐HT decreased Gαi3 activation.

In conclusion, at h5‐HT1B receptors expressed in CHO cells: (i) inverse agonist induced inhibition of Gαi3, and its reversal by S18127, reveals constitutive activation of this Gα subunit; (ii) constitutive Gαi3 activation can be quantified by isotopic dilution [35S]GTPγS binding and (iii) decreasing NaCl concentrations enhances Gαi3 activation and leads to protean agonist properties of 5‐HT: that is a switch to inhibition of Gαi3.

British Journal of Pharmacology (2003) 138, 1077–1084. doi:10.1038/sj.bjp.0705140

01 Nov 2000·Molecular PharmacologyQ3 · MEDICINE

Inverse Agonism and Constitutive Activity as Functional Correlates of Serotonin h5-HT_1BReceptor/G-Protein Stoichiometry

Q3 · MEDICINE

Article

Author: Verriele, Laurence ; Moreira, Celia ; Millan, Mark J. ; Audinot, Valerie ; Newman-Tancredi, Adrian

This study evaluated the influence of receptor/G-protein (R:G) stoichiometry on constitutive activity and the efficacy of agonists, partial agonists, and inverse agonists at human (h) 5-hydroxytryphamine 1B (5-HT(1B)) receptors. Two Chinese hamster ovary cell lines were used; they expressed 8.5 versus 0.4 pmol h5-HT(1B) receptors/mg (determined by [(3)H]GR125,743 saturation analysis) and 3.0 versus 1.5 pmol receptor-activated G-proteins/mg [determined by guanosine-5'-O-(3-[(35)S]thio)-triphosphate ([(35)S]GTPgammaS) isotopic dilution], respectively. Thus, they displayed R:G ratios of approximately 3.0 (RGhigh) and approximately 0.3 (RGlow), respectively. In competition-binding experiments, the agonists, 5-HT and sumatriptan, displayed fewer high-affinity (HA)-binding sites and the partial agonists, BMS181, 101 and L775,606, displayed decreased affinity in RGhigh versus RGlow membranes. In contrast, the inverse agonists, SB224,289 and, to a lesser extent, methiothepin, showed increased affinity. In G-protein activation experiments, both basal and 5-HT-activated [(35)S]GTPgammaS binding were higher in RGhigh than in RGlow membranes. Constitutive activity (determined by inhibition of basal [(35)S]GTPgammaS binding with GTPgammaS in the absence of receptor ligands) was more pronounced in RGhigh versus RGlow membranes, as revealed by the >5-fold greater proportion of HA sites. Correspondingly, the negative efficacy of inverse agonists was strikingly augmented, inasmuch as they suppressed approximately two-thirds of HA [(35)S]GTPgammaS binding in RGhigh membranes, but only approximately one-third in RGlow membranes. Furthermore, the efficacy of partial agonists was greater at RGhigh versus RGlow membranes, as estimated by their ability to enhance [(35)S]GTPgammaS binding. In conclusion, an increase in R:G ratios at h5-HT(1B) receptors was associated with an increase in relative efficacy of partial agonists and, most notably, an increase in both constitutive G-protein activation and negative efficacy of inverse agonists.

01 Oct 1999·Biomedical ChromatographyQ4 · MEDICINE

Sensitive triple-quadrupole mass spectrometric assay for the determination of BMS-181885, a 5-HT1 agonist, in human plasma following solid phase extraction

Q4 · MEDICINE

Article

Author: Shyu, Wen Chyi ; Srinivas, Nuggehally R. ; Whigan, Daisy ; Chang, Shu-Ying

A sensitive, selective, accurate, precise and reproducible triple-quadrupole liquid chromatographic-mass spectrometric assay was developed and validated for BMS-181885 (I), a 5HT1 agonist, in human plasma using BMS-181101 as the internal standard (IS). The method involved solid phase extraction of plasma containing I and the IS using Isoelute CN cartridges. The supernatant was then evaporated to dryness at 40 degrees C. The residue was dissolved in 100 microL of the injecting solvent. The HPLC column was ODS-3, 2 x 100 mm. The mobile phase comprised 10 mM ammonium formate (pH = 4) and acetonitrile, 55:45 v/v, used in an isocratic condition. The mass spectrometer was programmed to admit the protonated molecules at m/z 461 (I) and m/z 370 (IS) via the first quadrupole filter and to select reaction monitoring of ions at m/z 152 for I and IS for the quantification. Standard curves were fitted to a weighted quadratic function over the concentration range 0.2-200 ng/mL. The lowest standard concentration (0.2 ng/mL) was experimentally established as the lower limit of quantitation of the assay. The mean predicted quality control concentrations deviated within +/- 11% of the corresponding nominal values; the intra-assay and inter-assay precisions were within 7.0% relative standard deviation. I was stable in the injection solvent at 4 degrees C for at least 24 h and for at least three freeze-thaw cycles. Freezer stability of I in plasma was demonstrated for at least 3 months. The extraction recovery of I was established as 97%. The validated assay was applied to a pharmacokinetic study of I in humans.

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