Figure 5

Ca ²⁺ -entry evoked by pregnenolone sulphate (PregS) in primary cultures of FLSs from patients with RA. (a) Intracellular Ca²⁺ measurement showing the effect of external application of 100 μM PregS in the presence and absence of extracellular Ca²⁺. (b) As for (a) but the effect of PregS (100 μM) and then addition of 20 μM SPH. (c) As for (a) but paired experiments on the same 96-well plate where responses were compared after pre-treatment with anti-TRPM3 antibody (TM3E3) or TM3E3 preadsorbed to its antigenic peptide ("+pep."; control group). (a-c) All data points are N = 8 and representative of 3 (n) independent experiments. (d) Concentration-dependence of Ca²⁺-entry evoked by PregS. Mean data are shown for measurement of the peak and sustained responses to PregS (e.g. see (c)); 4 (n) independent experiments. The fitted curves are Hill equations with mid-points at 35.7 μM (peak) and 39.5 μM (sustained). The shaded area on the left (i) indicates the highest concentrations reported for endogenous PregS [22, 23], while the shaded area on the right (ii) indicates the higher concentrations of plasma PregS predicted on oral administration of 100 mg daily to adult humans.