Role of the nitric oxide/cyclic GMP pathway and extracellular environment in the nitric oxide donor-induced increase in dopamine secretion from PC12 cells: a microdialysis in vitro study Pier Andrea Serra,* Gaia Rocchitta,* Maria R. Delogu,* Rossana Migheli,* Maria G. Taras,* Maria P. Mura,* Giovanni Esposito,* Egidio Miele,* Maria S. Desole* and Maddalena Miele  *Department of Pharmacology, University of Sassari, Sassari, Italy  Maudsley Hospital, London, UK Abstract In vitro microdialysis was used to investigate the mechanism of nitric oxide (NO) donor-induced changes in dopamine (DA) secretion from PC12 cells. Infusion of the NO-donor S-nitroso- N-acetylpenicillamine (SNAP, 1.0 mM) induced a long-lasting increase in DA and 3-methoxytyramine (3-MT) dialysate concentrations. SNAP-induced increases were inhibited either by pre-infusion of the soluble guanylate cyclase (sGC) inhib- itor 1H-[1,2,4] oxadiazolo[4,3]quinoxalin-1-one (ODQ, 0.1 mM) or by Ca 2+ omission. Ca 2+ re-introduction restored SNAP ef- fects. SNAP-induced increases in DA + 3-MT were unaffected by co-infusion of the L-type Ca 2+ channel inhibitor nifedipine. The NO-donor (+ / –)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro- 3-hexenamide (NOR-3, 1.0 mM) induced a short-lasting decrease in dialysate DA + 3-MT. Ascorbic acid (0.2 mM) co-infusion allowed NOR-3 to increase dialysate DA + 3-MT. ODQ pre-infusion inhibited NOR-3 + ascorbic acid-induced DA + 3-MT increases. Infusion of high K + (75 mM) induced a 2.5-fold increase in dialysate DA + 3-MT. The increase was abolished by NOR-3 co-infusion. Conversely, co-infusion of ascorbic acid (0.2 mM) with NOR-3 + high K + restored high K + effects. Co-infusion of nifedipine inhibited high K + -induced DA + 3-MT increases. These results suggest that activation of the NO / sGC / cyclic GMP pathway may be the underlying mechanism of extracellular Ca 2+ -dependent effects of exogenous NO on DA secretion from PC12 cells. Extracellular Ca 2+ entry may occur through nifedipine-insensitive channels. NO effects and DA concentrations in dialysates largely depend on both the timing of NO generation and the extra- cellular environment in which NO is generated. Keywords: calcium, cyclic GMP, dopamine secretion, nitric oxide, PC12 cells. J. Neurochem. (2003) 86, 1403–1413. Nitric oxide (NO), a free radical gas, is a multifunctional and widespread biological messenger molecule. Roles proposed for NO in CNS pathophysiology are increasingly multiform and range from intracellular signaling (Garthwaite and Boulton 1995) to tissue damage or protection (Wink and Mitchell 1998; Murphy 2000). It is evident that either signaling, damaging or protective effects of NO depend on both the source of NO (neuronal, glial, extracellular, exogenous; Wink and Mitchell 1998; Murphy 2000) and the timing of NO generation (Yamamoto et al. 2000). In the mammalian brain, an increase in intracellular calcium following NMDA-mediated glutamatergic stimulation results in activation of NO synthase to form NO from the precursor, arginine (Kendrick et al. 1997). Activation of soluble guanylate cyclase (sGC) is one of the main intracellular effects of NO. The resulting increase in cyclic GMP levels modulates, among other activities, that of ion channels (Knowles et al. 1989). The NO / cyclic GMP pathway plays an important role in Ca 2+ cycling. Cyclic GMP may either Received February 10, 2003; revised manuscript received April 26, 2003; accepted May 22, 2003. Address correspondence and reprint requests to M. S. Desole, Department of Pharmacology, Faculty of Medicine, University of Sassari, Viale S. Pietro 43B, 07100 Sassari, Italy. E-mail: Pharmaco@ssmain.uniss.it Abbreviations used: DA, dopamine; DHAA, dehydroascorbic acid; DOPAC, dihydroxyphenylacetic acid; DMEM, Dulbecco’s modified Eagle’s medium F12; HVA, homovanillic acid; 3-MT, 3-methoxytyr- amine; ODQ, 1H-[1,2,4] oxadiazolo[4,3]quinoxalin-1-one; NOR-3, (+ / –)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide; OPD, O-phenylendiamine; PBS, phosphate-buffered saline; sGC, soluble guanylate cyclase; SNAP, S-nitroso-N-acetylpenicillamine. Journal of Neurochemistry, 2003, 86, 1403–1413 doi:10.1046/j.1471-4159.2003.01947.x Ó 2003 International Society for Neurochemistry, J. Neurochem. (2003) 86, 1403–1413 1403