Characterisation of parathyroid hormone receptors (PTH-R) present in human SaOS2 osteoblast-like cells

D. Sykes, E.A.Harper, W. Xun, M. Shaxted, E. P. Griffin, S. Craib, D. Patel, M. Raynor, I. McDonald, C. Austin, R.A.D. Hull, & S. B. Kalindjian. James Black Foundation, Dulwich, London, UK.

The human PTH1-R and PTH2-R are both coupled to Gs and their activation leads to an increase in intracellular cAMP (Hoare & Usdin 2001). We have already developed PTH1-R assays to determine the affinity and efficacy of novel chemical entities (NCE’s) at human cloned PTH1-Rs. However, we also wished to measure ligand affinity and efficacy at endogenous human PTH1-Rs. Here, we have used a LANCETM cAMP assay (PerkinElmer), the PTH receptor agonist, PTH(1-34), the PTH1-R-selective agonist, PTHrP(1-34), the PTH2-R-selective agonist, TIP(1-39), and the PTH-R antagonist, [Nle30] tuberoinfundibular peptide(7-39) (NleTIP), to characterise human PTH receptors present in SaOS2 cells.

Frozen SaOS2 cells were thawed into RPMI containing 25mM HEPES (pH7.4, 21ºC), 0.5mg ml-1 bacitracin, 2.5mM IBMX and 1mM rolipram. To determine whether PTH-R ligands were agonists in SaOS2 cells, they were incubated (5 μl, 100 μM–100 μM) with cells (5 μl, 3x103), in 384 well Optiplates for 120min. Detection mix containing Alexa Fluor ® 647-anti cAMP antibody, europium-labelled streptavidin, biotinylated cAMP, 0.5% BSA and 2.5mM IBMX was added and after 24h the fluorescence at 665nm measured on an EnVision. Antagonist affinity was determined by preincubion of cells for 30min before addition to 384-well Optiplates containing PTH-R agonists, incubation for 120min and subsequent detection of cAMP.

Both PTH(1-34) and PTHrP(1-34) (10pM-1μM) produced an increase in cAMP accumulation although the pEC50 of PTHrP(1-34) was significantly higher (paired t-test, p<0.05; see table 1, mean ± s.e.mean ).

TIP(1-39) and NleTIP (10pM – 10 μM) did not induce cAMP accumulation (table1). NleTIP (3-300nM) produced concentration-dependent parallel rightward shifts in PTH(1-34) and PTHrP(1-34) concentration effect (E/[A]) curves with no change in maximal response ( α). Schild analysis of 6 PTH(1-34) experiments and 4 PTHrP(1-34) experiments demonstrates that NleTIP is a simple competitive antagonist at PTH-R in SaOS2 cells. The data was best fit by the Schild equation with unit slope (p<0.05, F-test), giving pKB values of 8.35 ± 0.19 (b = 0.96 ± 0.12; vs PTH(1-34)) and 8.46 ± 0.17 (b = 0.92 ± 0.15; vs PTHrP(1-34)). TIP(1-39) (300nM) produced a parallel rightward shift in PTH(1-34) E/[A] curves giving a pA2 value of 7.12 ± 0.07 (n = 4 ± s.e.mean).

In conclusion, the rank order of potency of PTH-R ligands and the agonist independent affinity of NleTIP indicate that SaOS2 cells express PTH1-receptors but not PTH2-receptors. SaOS2 cells and a LANCETM-based cAMP assay can be used for determining the affinity and efficacy of NCE’s at human endogenous PTH1-receptors.

Hoare S. R. J. &. Usdin T. B. (2001) Current Pharmaceutical Design 7, 689-713.