Nitric oxide induces muscular relaxation via cyclic GMP-dependent and -independent mechanisms in the longitudinal muscle of the mouse duodenum Rosa Serio, * Maria Grazia Zizzo, and Flavia Mul  e Dipartimento di Biologia Cellulare e dello Sviluppo, Laboratorio di Fisiologia Generale, Universit  a di Palermo, 90128 Palermo, Italy Received 17 July 2002 Abstract The aim of this study was to investigate, in mouse duodenum, the role of nitric oxide (NO) in the relaxation of longitudinal muscle evoked by nerve activation and the coupled action mechanism. Electrical field stimulation (EFS; 0.5 ms, 10-s train duration, supramaximal voltage, at various frequencies) under nonadrenergic noncholinergic conditions evoked muscular relaxation occa- sionally followed, at the higher stimulus frequencies, by rebound contractions. Inhibition of the synthesis of NO by N x -nitro-L - arginine methyl ester (L -NAME; 100 lM) virtually abolished the evoked relaxation. The relaxation was reduced also by apamin (0.1 lM) and by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 1 lM), a guanylyl cyclase inhibitor. The coadministration of apamin and ODQ produced additive effects on the responses to EFS. Sodium nitroprusside (0.1–100 lM) produced a concentration- dependent reduction of the phasic spontaneous activity and at the highest dose used suppressed phasic activity and induced muscular relaxation. These effects were tetrodotoxin and L -NAME resistant and were antagonized both by apamin and by ODQ. 8-Bromoguanosine 3 0 ,5 0 -cyclic monophosphate (0.1–100 lM) reduced in a concentration-dependent manner the spontaneous me- chanical activity and at 100 lM suppressed the phasic activity and induced muscular relaxation, not antagonized by apamin. This study indicates that NO is the primary transmitter released by inhibitory nerves supplying the longitudinal muscle of mouse du- odenum and that guanylate cyclase stimulation and opening of Ca 2þ -dependent K þ channels are independent mechanisms working in parallel to mediate NO action. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Nonadrenergic noncholinergic relaxation; Nitric oxide; Cyclic GMP; K þ -channels; Mouse duodenum The release of inhibitory neurotransmitters from nonadrenergic noncholinergic (NANC) 1 neurons medi- ates many gastrointestinal motility patterns and is re- sponsible for maintenance of gastrointestinal muscle in a state of inhibition. ATP or related purines, vasoactive intestinal peptide, and, more recently, nitric oxide (NO) and pituitary adenylate cyclase activating peptide have been suggested to be involved in NANC inhibitory transmission in the enteric nervous system [1]. Their relative contributions vary between gastrointestinal re- gions and between mammalian species however, NO seems to be the main inhibitory transmitter at most neuromuscular junctions. Many of the actions of NO involve the guanosine 3 0 ,5 0 -cyclic monophosphate (cGMP) generating system. Production of cGMP and, perhaps, phosphorylation of cellular proteins transduce the NO signal to induce re- laxation of smooth muscle [2]. However, in a variety of tissues NO can have other targets: NO can act through ion channels [3,4], increasing K þ conductance, which induces hyperpolarization of cell membrane, or activing tyrosine kinases, phosphatases [5], GTPases [6], cyto- solic ADP ribosyltransferases [7], or transcription fac- tors [5]. Nitric Oxide 8 (2003) 48–52 www.elsevier.com/locate/yniox NITRIC OXIDE Biology and Chemistry * Corresponding author. Fax: +39-091-6577501. E-mail address: rserio@unipa.it (R. Serio). 1 Abbreviations used: 8-Br-cGMP, 8-bromoguanosine 3 0 ;5 0 -cyclic monophosphate; cGMP, guanosine 3 0 ,5 0 -cyclic monophosphate; EFS, electrical field stimulation; L-NAME, N x -nitro-L-arginine methyl ester; NANC, nonadrenergic noncholinergic; NO, nitric oxide; NOS, nitric oxide synthase; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; SNP, sodium nitroprusside; TTX, tetrodotoxin. 1089-8603/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII:S1089-8603(02)00144-1