The nitric oxide/cGMP pathway couples muscarinic receptors to the activation of Ca2+ influx.
Inward currents activated by 8-bromc-cGMP and by muscarinic agonist were compared in N1E-115 mouse neuroblastoma cells using perforated-patch voltage clamp and Fura-2 imaging. The cGMP analog activates a voltage-independent inward current that is carried at least in part by Ca2+ because it persists in Na(+)-free saline when Ca2+ is present and is blocked by external Mn2+ and Ba2+. The current is similar to the inward current that develops during stimulation of M1 muscarinic receptors, and the currents activated by agonist and by 8-bromo-cGMP are not additive, indicating that the same pathway is involved. Inhibition of cGMP production with NG-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of nitric oxide (NO)-synthase, prevents activation of Ca2+ current by agonist without affecting the content of intracellular Ca2+ stores or the ability of agonist to mobilize Ca2+. The inhibition is overcome by 8-bromo-cGMP. LY83583, a competitive inhibitor of guanylyl cyclase, reversibly blocks activation of Ca2+ current by agonist, again without affecting the content of Ca2+ stores or Ca2+ release. Rp-8-pCPT-cGMPS, 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 linking muscarinic receptor occupancy to the activation of voltage-independent Ca2+ current. The pathway involves positive feedback. Calcium entering via voltage-independent channels preferentially stimulates NO-synthase, which leads to enhanced cGMP production and greater Ca2+ influx. Positive feedback may explain the rapid increase in cGMP that occurs during muscarinic receptor activation.
Mathes
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Department of Biological Sciences, Stanford University, Pacific Grove, California 93950, USA.
Thompson
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D000818:Animals; D002118:Calcium / Q000378:metabolism*; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors; D051379:Mice; D018721:Muscarinic Agonists / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D011976:Receptors, Muscarinic / Q000378:metabolism*; D013873:Thionucleotides / Q000494:pharmacology; D014407:Tumor Cells, Cultured
C090860:8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphorothioate; D018721:Muscarinic Agonists; D011976:Receptors, Muscarinic; D013873:Thionucleotides; C016276:8-bromocyclic GMP; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D006162:Guanylate Cyclase; D006152:Cyclic GMP; D002118:Calcium