Inhibitors of arachidonate metabolism and perturbants of the oxidation-reduction state of the cell were employed to develop a pharmacologic profile for muscarinic receptor-mediated cyclic GMP formation in murine neuroblastoma cells (clone N1E-115). Several lipoxygenase inhibitors [eicosatetraynoic add (ETYA), nordihydroguaiaretic acid (NDGA), FPL 57231, FPL 55712, BW755c, propylgallate, and AA861] blocked the elevation of [3H]cyclic GMP induced by muscarinic receptor activation. The cyclooxygenase inhibitors indomethacin and ibuprofen were two orders of magnitude less potent in blocking the muscarinic receptor-mediated [3H]cyclic GMP response than in blocking cyclooxygenase in other systems. ETYA and NDGA did not affect the muscarinic inhibition of the prostaglandin E1-mediated increases in [3H]cyclic AMP levels in N1E-115 cells. ETYA did not have a reproducible effect on the muscarinic receptor-induced release of inositol phosphates. Thus, these lipoxygenase inhibitors appeared to be selective for the effector system coupled to the low-affinity muscarinic agonist-receptor conformation, i.e. that which induces cyclic GMP formation. Other effective inhibitors of the cyclic GMP response were methylene blue, catalase, bromphenacyi bromide, retinal, dithiothreitol, quinacrine, and oxidized glutathione. The antioxidant α-tocopherol in the concentration range of 100 μM to 1 mM potentiated the receptor response. Arachidonic acid itself was an inhibitor of the muscarinic receptor-mediated cyclic GMP response (ic50 = 45 μM). Linoleic acid and oleic acid were less potent (ic50 = 130 and 190μM, respectively), and stearic acid was ineffective. When arachidonic acid was air-oxidized, its inhibitory potency was increased 10-fold. Most but not all of the spontaneously-produced oxidative metabolites, separable by reverse-phase high pressure liquid chromatography, were inhibitory to the receptor response. Enzymatically synthesized 12-hydroxyeicosatetraenoic add and 15-hydroxyeicosatetraenoic add inhibited the muscarinic receptor [3H]cyclic GMP response, with ic50 values of 17 and 8 μM respectively. Catalase was effective in blocking the muscarinic cyclic GMP response (ic50 = 5μM) while having no effect on either the muscarinic receptor-induced inositol phosphate release or the reduction of cyclic AMP levels. Thus, the effector system for increasing cyclic GMP in these cells displays many of the expected characteristics for the involvement of a lipoxygenase or a related enzyme that oxidatively metabolizes arachidonate in order to activate the guanylate cyclase. A number of pharmacologic agents differentiate between this effector system and those to which the muscarinic receptor couples in order to effect cyclic AMP decreases or the release of inositol phosphates.
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