Abstract

Results of previous pharmacological studies suggested that presynaptic muscarinic M₂ receptors on cerebral perivascular nitric oxidergic (nitrergic) nerves mediated inhibition of nitric oxide release from these nerves. The inhibition was thought to be primarily attributable to a decreased Ca²⁺ influx through N-type Ca²⁺ channels on nitrergic nerves, but direct evidence supporting this hypothesis was not presented. In the present study, we used cultured rat sphenopalatine ganglion (SPG), a major source of nitrergic nerves to cerebral blood vessels, to investigate the role of muscarinic M₂ receptors in modulating voltage-dependent Ca²⁺ channels. SPG neuronal soma and dendrites were immunoreactive for both N-type Ca²⁺ channels and muscarinic M₂ receptors, indicating that muscarinic M₂receptors were colocalized with N-type Ca²⁺ channels. Using the whole-cell voltage-clamp technique, we found that voltage-dependent Ca²⁺ currents in cultured SPG were largely blocked by ω-conotoxin, an N-type calcium channel antagonist, but were not affected by nifedipine, an L-type calcium antagonist. The Ca²⁺ current was inhibited by acetylcholine (ACh) and arecaidine but-2-ynyl ester tosylate (ABET), a preferential muscarinic M₂-receptor agonist, in a concentration-dependent manner. The inhibition was reversed by atropine and methoctramine (a muscarinic M₂-receptor antagonist), but was not affected by muscarinic M₁-, M₃-, or M₄-receptor antagonists. Consistent with this, preferential muscarinic M₁-receptor agonists McN-A-343 and oxotremorine did not affect the Ca²⁺ current. Furthermore, pretreatment with pertussis toxin and guanosine 5′-O-(3-thio)triphosphate prevented ACh and ABET inhibition of Ca²⁺ currents. These results are consistent with pharmacological findings in the pig basilar arteries and provide direct evidence supporting our hypothesis that M₂-receptor-mediated inhibition of cerebral nitrergic neurogenic vasodilation is due to a G_i-protein-mediated suppression of Ca²⁺ influx via voltage-dependent N-type Ca²⁺ channels on perivascular nerves.