Roles of arachidonic acid, lipoxygenases and phosphatases in calcium-dependent modulation of M-current in bullfrog sympathetic neurons.
1. M-current (IM) is regulated by intracellular free Ca2+ ([Ca2+]i). Suppression and overrecovery of IM induced by muscarine and luteinizing-hormone releasing hormone (LHRH) are also regulated by [Ca2+]i. The role of the arachidonic acid (AA) pathway in the Ca(2+)-dependent modulation of IM was investigated using whole-cell voltage clamp and intracellular perfusion in dissociated bullfrog sympathetic B neurons. 2. Quinacrine (10-20 microM) and 4-bromophenacyl bromide (4-BPB; 4-10 microM), the inhibitors of phospholipase A2, blocked the enhancement of IM evoked by raising [Ca2+]i. 3. AA (6-120 microM) increased IM by about 50% of the control current in a Ca(2+)-dependent manner. 4. Enhancements of IM by Ca2+ and AA were blocked by the lipoxygenase (LO) inhibitors nordihydroguaiaretic acid (NDGA; 1-5 microM) and 5,8,11-eicosatrynoic acid (ETI; 10 microM). The cyclo-oxygenase inhibitor indomethacin (10 microM) had no effect. 5. Enhancement of IM by Ca2+ was abolished by the selective 12-LO inhibitors baicalein (1-2 microM) and 15(S)-hydroxy-5-cis-8-cis-11-cis-13-trans-eicosatetraenoic acid (15-HETE; 6.5 microM). A 12-LO product, 2(S)-hydroxy-5-cis-8-cis-10-trans-14-cis- eicosatetraenoic acid (12-HETE; 13-20 microM), increased IM without Ca2+ requirement. 6. Enhancement of IM by Ca2+ was not affected by the selective 5-LO inhibitors AA-861 (10 microM), 5,6-dehydroarachidonic acid (5,6-DAA, 10 microM) and L-651,896 (10 microM). The 5-LO metabolites leukotriene C4 (1.5-8 microM) and leukotriene B4 (1.5-5 microM) showed no obvious effect on IM. 7. NDGA alone inhibited IM with an IC50 of 0.73 microM at 120 nM Cai(2+). 8. NDGA did not affect suppression of IM by muscarine or LHRH; however, overrecovery of IM upon removing these agonists was totally eliminated by 1 microM NDGA. 9. Inhibitors of phosphatases, calyculin A (0.1 microM) and okadaic acid (1 microM), completely abolished overrecovery of IM. Calyculin A also blocked the Ca(2+)-induced IM enhancement. 10. It is suggested that Ca2+ enhances IM by stimulating the AA metabolic pathway. Dephosphorylation probably upregulates IM. Overrecovery of IM is probably a result of stimulation of the LO pathway and phosphatases by increased [Ca2+]i.
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Howard Hughes Medical Institute, Department of Neurobiology and Behavior, State University of New York at Stony Brook 11794, USA.
D000818:Animals; D016718:Arachidonic Acid / Q000502:physiology; D002118:Calcium / Q000502:physiology*; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism / Q000502:physiology; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D005728:Ganglia, Sympathetic / Q000166:cytology / Q000201:enzymology / Q000502:physiology; D066298:In Vitro Techniques; D008084:Lipoxygenase / Q000502:physiology; D016859:Lipoxygenase Inhibitors / Q000494:pharmacology; D009474:Neurons / Q000201:enzymology / Q000502:physiology*; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000502:physiology; D015221:Potassium Channels / Q000502:physiology*; D011892:Rana catesbeiana; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000502:physiology; D013564:Sympathetic Nervous System / Q000166:cytology / Q000201:enzymology / Q000502:physiology*
D016861:Cyclooxygenase Inhibitors; D016859:Lipoxygenase Inhibitors; D015221:Potassium Channels; D011976:Receptors, Muscarinic; D016718:Arachidonic Acid; D008084:Lipoxygenase; D010744:Phosphoric Monoester Hydrolases; D002118:Calcium