title: Presynaptic muscarinic M(2)-receptor-mediated inhibition of N-type Ca(2+) channels in cultured sphenopalatine ganglion: direct evidence for acetylcholine inhibition of cerebral nitrergic neurogenic vasodilation.
abstract: 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(2+) influx through N-type Ca(2+) 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(2+) channels. SPG neuronal soma and dendrites were immunoreactive for both N-type Ca(2+) channels and muscarinic M(2) receptors, indicating that muscarinic M(2) receptors were colocalized with N-type Ca(2+) channels. Using the whole-cell voltage-clamp technique, we found that voltage-dependent Ca(2+) currents in cultured SPG were largely blocked by omega-conotoxin, an N-type calcium channel antagonist, but were not affected by nifedipine, an L-type calcium antagonist. The Ca(2+) 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(2+) current. Furthermore, pretreatment with pertussis toxin and guanosine 5'-O-(3-thio)triphosphate prevented ACh and ABET inhibition of Ca(2+) currents. These results are consistent with pharmacological findings in the pig basilar arteries and provide direct evidence supporting our hypothesis that M(2)-receptor-mediated inhibition of cerebral nitrergic neurogenic vasodilation is due to a G(i)-protein-mediated suppression of Ca(2+) influx via voltage-dependent N-type Ca(2+) channels on perivascular nerves.
authors.affiliation: Department of Pharmacology, Southern Illinois University School of Medicine, Springfield, IL 62794-9629, USA.
authors.forename: J
authors.initials: J
authors.lastname: Liu
authors.forename: M S
authors.initials: MS
authors.lastname: Evans
authors.forename: T J-F
authors.initials: TJ
authors.lastname: Lee
mesh_terms: D015124:8-Bromo Cyclic Adenosine Monophosphate; D000109:Acetylcholine / Q000494:pharmacology*; D000818:Animals; D001488:Basilar Artery / Q000187:drug effects / Q000294:innervation / Q000502:physiology; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D020864:Calcium Channels, N-Type / Q000187:drug effects*; D002478:Cells, Cultured; D002560:Cerebrovascular Circulation / Q000187:drug effects*; D018678:Cholinergic Agents / Q000494:pharmacology; D004594:Electrophysiology; D005726:Ganglia, Parasympathetic / Q000187:drug effects*; D007150:Immunohistochemistry; D008297:Male; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D009569:Nitric Oxide / Q000037:antagonists & inhibitors* / Q000502:physiology; D009924:Organ Culture Techniques; D018408:Patch-Clamp Techniques; D037342:Pertussis Toxin; D051381:Rats; D017207:Rats, Sprague-Dawley; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic / Q000187:drug effects*; D017661:Receptors, Presynaptic / Q000187:drug effects*; D013552:Swine; D014664:Vasodilation / Q000187:drug effects / Q000502:physiology*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D002121:Calcium Channel Blockers; D020864:Calcium Channels, N-Type; D018678:Cholinergic Agents; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic; D017661:Receptors, Presynaptic; D010566:Virulence Factors, Bordetella; D015124:8-Bromo Cyclic Adenosine Monophosphate; D009569:Nitric Oxide; D037342:Pertussis Toxin; D000109:Acetylcholine