BML-241 fails to display selective antagonism at the sphingosine-1-phosphate receptor, S1P3

Background and purpose: The thiazolidine carboxylic acid, BML-241, has been proposed as a lead compound in development of selective antagonists at the sphingosine-1-phosphate receptor (S1P(3)), based on its inhibition of the rise in intracellular calcium concentrations ([Ca2+](i)) in HeLa cells overexpressing S1P receptors. We have studied the antagonistic properties of BML-241 for the S1P3 receptor in more detail. Experimental approach: Chinese hamster ovary (CHO) cells stably transfected with the S1P(3), S1P(2) or alpha(1A)-adrenoceptors were used to investigate the effect of BML-241 on increases in [Ca2+](i) mediated via different receptors. CHO-K1 cells were used to study ATP-induced [Ca2+](i) elevations. Effects on S1P(3)-mediated inhibition of forskolin-induced cAMP accumulation and on binding to alpha(1A)-adrenoceptors were also investigated. In addition, the effect of BML-241 on contractions of rat mesenteric artery induced by phenylephrine was studied in an organ bath. Key results: High concentrations of BML-241 (10 mu M) inhibited the rise in [Ca2+](i) induced by S1P(3) and S1P(2) receptor stimulation; lower concentrations were ineffective. This high concentration of BML-241 also inhibited [Ca2+](i) increases via P2 (nucleotide) receptor or alpha(1A)-adrenoceptor stimulation. Moreover, BML-241 (10 mM) inhibited alpha(1A)-adrenoceptor-mediated contraction of rat mesenteric artery but did not displace [H-3]-prazosin from alpha(1A)-adrenoceptors in concentrations up to 100 mM. BML-241 (10 mu M) did not affect the S1P(3)-mediated decrease of forskolin-induced cAMP accumulation. Conclusions and Implications: We conclude that BML-241 is a low potency, non-selective inhibitor of increases in [Ca2+](i), rather than a specific antagonist at the S1P(3) receptor.