Nitric oxide (NO) is a major transmitter in mediating cerebral neurogenic vasodilation in several species. Recent findings have suggested that acetylcholine, which is costored with NO in cerebral perivascular nerves, plays a role in modulating NO release, presumably by acting on muscarinic (M) receptors on nitric oxidergic nerve terminals. The present study was designed using an in vitro tissue bath technique to pharmacologically characterize the presynaptic muscarinic-receptor subtype(s) that mediate modulation of NO release and therefore neurogenic vasodilation and to investigate further the possible mechanisms involved in this presynaptic modulation in porcine basilar arteries. Transmural nerve stimulation (TNS) elicited a frequency-dependent, tetrodotoxin-sensitive relaxation. The relaxation was abolished by nitro-l-arginine (30 μM) and was completely reversed byl-arginine andl-citrulline, but not by their d-enantiomers. Atropine (0.01–1 μM), pirenzepine (an M1-receptor antagonist, 0.01–1 μM), and methoctramine (an M2-receptor antagonist, 0.01–1 μM), but not 4-DAMP (an M3-receptor antagonist) or tropicamide (an M4-receptor antagonist) at concentrations as high as 10 mM, significantly increased the TNS-elicited relaxation. This relaxation, on the other hand, was significantly attenuated by arecaidine but-2-ynyl ester tosylate (an M2-receptor agonist, 0.1 μM) but was not affected by McN-A-343 (an M1-receptor agonist, 1 μM). Double-labeling immunohistochemical study demonstrated that perivascular M2receptor-immunoreactive fibers were completely coincident with NADPH diaphorase fibers. Furthermore, the muscarinic receptor-mediated modulation of TNS-elicited relaxation was completely prevented by ω-conotoxin GVIA (0.1 μM), a specific N-type Ca2+ channel inhibitor, but was still observed in the presence of tetraethylammonium (1 mM), 8-bromo-cAMP (0.5 mM), and pertussis toxin. It is concluded that the presynaptic M2 receptors on porcine cerebral perivascular nitric oxidergic nerves mediate inhibition of NO release. The inhibition is due primarily to a decreased Ca2+ influx through N-type Ca2+ channels.

Results of previous pharmacological studies suggested that presynaptic muscarinic M 2 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 Ca 2ϩ influx through N-type Ca 2ϩ 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 M 2 receptors in modulating voltage-dependent Ca 2ϩ channels. SPG neuronal soma and dendrites were immunoreactive for both N-type Ca 2ϩ channels and muscarinic M 2 receptors, indicating that muscarinic M 2 receptors were colocalized with N-type Ca 2ϩ channels. Using the whole-cell voltage-clamp technique, we found that voltagedependent Ca 2ϩ currents in cultured SPG were largely blocked by -conotoxin, an N-type calcium channel antagonist, but were not affected by nifedipine, an L-type calcium antagonist.The Ca 2ϩ current was inhibited by acetylcholine (ACh) and arecaidine but-2-ynyl ester tosylate (ABET), a preferential muscarinic M 2 -receptor agonist, in a concentration-dependent manner. The inhibition was reversed by atropine and methoctramine (a muscarinic M 2 -receptor antagonist), but was not affected by muscarinic M 1 -, M 3 -, or M 4 -receptor antagonists. Consistent with this, preferential muscarinic M 1 -receptor agonists McN-A-343 and oxotremorine did not affect the Ca 2ϩ current. Furthermore, pretreatment with pertussis toxin and guanosine 5Ј-O-(3-thio)triphosphate prevented ACh and ABET inhibition of Ca 2ϩ currents. These results are consistent with pharmacological findings in the pig basilar arteries and provide direct evidence supporting our hypothesis that M 2 -receptormediated inhibition of cerebral nitrergic neurogenic vasodilation is due to a G i -protein-mediated suppression of Ca 2ϩ influx via voltage-dependent N-type Ca 2ϩ channels on perivascular nerves.