S1P-receptors in PC12 and transfected HEK293 cells: molecular targets of hypotensive imidazoline I(1) receptor ligands

The present study aimed at elucidating the molecular identity of the proposed “I₁-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 [³H]clonidine and [³H]lysophosphatidic acid on intact, 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 S1P₁-, S1P₂- or S1P₃-receptor abolished specific [³H]lysophosphatidic acid binding. [³H]Clonidine binding was markedly decreased by siRNA targeting S1P₁- and S1P₃-receptors but not by siRNA against S1P₂-receptors. Finally, in HEK293 cells transiently expressing human S1P₃-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 “I₁-imidazoline receptors”.

The present results indicate that the “I₁-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 I₁ imidazoline receptors represent a mixture of S1P₁- and S1P₃-receptors and/or hetero-dimers of both.     

Modulation of N-type Ca currents by moxonidine via imidazoline I1 receptor activation in rat superior cervical ganglion neurons

Moxonidine, an imidazoline deriviatives, suppress the vasopressor sympathetic outflow to produce hypotension. This effect has been known to be mediated in part by suppressing sympathetic outflow via acting imidazoline I₁ receptors (IR₁) at postganglionic sympathetic neurons. But, the cellular mechanism of IR₁-induced inhibition of noradrenaline (NA) release is still unknown. We therefore, investigated the effect of IR₁ activation on voltage-dependent Ca²⁺ channels which is known to play an pivotal role in regulating NA in rat superior cervical ganglion (SCG) neurons, using the conventional whole-cell patch-clamp method. In the presence of rauwolscine (3 μΜ), which blocks α₂-adrenoceptor (R_α2), moxonidine inhibited voltage-dependent Ca²⁺ current (I_Ca) by about 30%. This moxonidine-induced inhibition was almost completely prevented by efaroxan (10 μΜ) which blocks IR₁ as well as R_α2. In addition, ω-conotoxin (CgTx) GVIA (1 μΜ) occluded moxonidine-induced inhibition of I_Ca, but, moxonidine-induced I_Ca inhibition was not affected by pertussis toxin (PTX) nor shows any characteristics of voltage-dependent inhibition. These data suggest that moxonidine inhibit voltage-dependent N-type Ca²⁺ current (I_Ca–N) via activating IR₁. Finally, moxonidine significantly decreased the frequency of AP firing in a partially reversible manner. This inhibition of AP firing was almost completely occluded in the presence of ω-CgTx. Taken together, our results suggest that activation of IR₁ in SCG neurons reduced I_Ca–N in a PTX-and voltage-insensitive pathway, and this inhibition attenuated repetitive AP firing in SCG neurons.