Clonidine and Cirazoline Inhibit Activation of Nicotinic Channels in PC‐12 Cellsfn1

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Abstract
Clonidine and cirazoline bind with high affinity to a nonadrenergic site in the brain stem, the so-called imidazoline I1 receptor. Our aim was to determine the mechanism by which these receptors act and their possible linkage to signal-transducing heterotrimeric G-proteins. We examined the effects of clonidine and cirazoline on PC-12 cells, a neuronal cell line that is reported to possess the I1 site and have no alpha 2-adrenoceptors. In undifferentiated PC-12 cells loaded with the Ca2+ indicator dye fura-2, clonidine and cirazoline (10-100 microM) inhibited the increase in [Ca2+]i produced by nicotine (10 microM). This inhibition was not reversed by yohimbine (100 microM), and adrenaline and BHT 920 were ineffective at 100 microM. This effect was not inhibited by pretreatment with pertussis toxin (24 hours, 100 ng/ml) and not modulated by pretreatment with IBMX (100 microM). The nicotine-induced increase in [Ca2+]i is apparently due to Ca2+ entering via the intrinsic ion channel of the nicotinic acetylcholine receptor. Clonidine and cirazoline inhibited the inward current produced by nicotine (10 microM) as measured by the whole cell patch-clamp technique in differentiated PC-12 cells, recorded at a holding potential of -60 mV. In agreement with the results found with fura-2, inhibition of inward current was concentration dependent and not blocked by yohimbine (100 microM) or mimicked by adrenaline (100 microM). Pretreatment of PC-12 cells with pertussis toxin or infusion of GDP-beta-S (2 mM) via the patch pipette did not alter the inhibition of the nicotine-induced inward current by clonidine or cirazoline. Clonidine and cirazoline, but not adrenaline, displayed [3H]phencyclidine from Torpedo electroplaque membranes enriched in nicotinic acetylcholine receptors in a concentration-dependent manner (10-100 microM). Taken together, these results suggest that clonidine and cirazoline inhibit Na+ and Ca2+ entry through the nicotinic acetylcholine receptor via a nonadrenergic mechanism that is independent of G-proteins and cyclic nucleotides, presumably by direct blockade of the intrinsic ion channel of the nicotinic acetylcholine receptor.