THE NITRIC OXIDE CGMP PATHWAY COUPLES MUSCARINIC RECEPTORS TO THE ACTIVATION OF CA2+ INFLUX/

Inward currents activated by 8-bromo-cGMP and by muscarinic agonist we re compared in N1E-115 mouse neuroblastoma cells using perforated-patc h voltage clamp and Fura-2 imaging. The cGMP analog activates a voltag e-independent inward current that is carried at least in part by Ca2because it persists in Na+-free saline when Ca2+ is present and is blo cked by external Mn2+ and Ba2+. The current is similar to the inward c urrent that develops during stimulation of M1 muscarinic receptors, an d the currents activated by agonist and by 8-bromo-cGMP are not additi ve, indicating that the same pathway is involved. Inhibition of cGMP p roduction with N-G-monomethyl-L-arginine (L-NMMA), a competitive inhib itor of nitric oxide (NO)-synthase, prevents activation of Ca2+ curren t by agonist without affecting the content of intracellular Ca2+ store s or the ability of agonist to mobilize Ca2+. The inhibition is overco me by 8-bromo-cGMP, LY83583, a competitive inhibitor of guanylyl cycla se, reversibly blocks activation of Ca2+ current by agonist, again wit hout affecting the content of Ca2+ stores or Ca2+ release. Rp-8-pCPT-c GMPS, an inhibitory analog of cGMP, also reduces the Ca2+ current and reduces Ca2+ influx during muscarinic activation, It is concluded that cGMP is the necessary and sufficient intermediate in the pathway link ing muscarinic receptor occupancy to the activation of voltage-indepen dent Ca2+ current. The pathway involves positive feedback. Calcium ent ering via voltage-independent channels preferentially stimulates NO-sy nthase, which leads to enhanced cGMP production and greater Ca2+ influ x. Positive feedback may explain the rapid increase in cGMP that occur s during muscarinic receptor activation.