S1P-receptors in PC12 and transfected HEK293 cells: Molecular targets of hypotensive imidazoline I1 receptor ligands

The present study aimed at elucidating the molecular identity of the proposed “I 1 -imidazoline receptors”, i.e. non-adrenoceptor recognition sites via which the centrally acting imidazolines clonidine and moxonidine mediate a major part of their effects. In radioligand binding experiments with [ 3 H]clonidine and [ 3 H]lysophosphatidic acid on intact, α 2 -adrenoceptor-deficient PC12 cells, moxonidine, clonidine, lysophosphatidic acid and sphingosine-1-phosphate (S1P) competed for the specific binding sites of both radioligands with similar affinities. RNA interference with the rat S1P 1 -, S1P 2 - or S1P 3 -receptor abolished specific [ 3 H]lysophosphatidic acid binding. [ 3 H]Clonidine binding was markedly decreased by siRNA targeting S1P 1 - and S1P 3 -receptors but not by siRNA against S1P 2 -receptors. Finally, in HEK293 cells transiently expressing human S1P 3 -receptors, sphingosine-1-phosphate, clonidine and moxonidine induced increases in intracellular calcium concentration, moxonidine being more potent than clonidine; this is in agreement with the known properties of the “I 1 -imidazoline receptors”. The present results indicate that the “I 1 -imidazoline receptors” mediating effects of clonidine and moxonidine in PC12 and the transfected HEK293 cells belong to the S1P-receptor family; in particular, the data obtained in PC12 cells suggest that the I 1 imidazoline receptors represent a mixture of S1P 1 - and S1P 3 -receptors and/or hetero-dimers of both.