Roles of arachidonic acid, lipoxygenases and phosphatases in calcium‐dependent modulation of M‐current in bullfrog sympathetic neurons.

Abstract

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. © 1995 The Physiological Society