Nested reverse transcription-polymerase chain reaction (RT-PCR) and 5′ and 3′ rapid amplification of cDNA ends (RACE) PCR generated a full-length cDNA sequence (3982·bp) of land crab NO synthase (Gl-NOS) from molting gland (Y-organ) and thoracic ganglion

Nitric oxide (NO) appears to have evolved as a signaling molecule before the radiation of the metazoans (Feelisch and Martin, 1995; Torreilles, 2001). NO is generated by nitric oxide synthase (NOS) from L-arginine, O2 and NADPH and diffuses freely across the cell membrane to induce responses in neighboring cells (Colasanti and Venturini, 2000). The bestknown NO signaling pathway is one in which NO activates a soluble class I guanylyl cyclase (GC-I; Baranano and Snyder, 2001). Activated GC-I produces cyclic 3′,5′-guanosine monophosphate (cGMP), which in turn activates cGMPdependent protein kinase. In mammals, NO/cGMP signaling is involved in vasodilation, neurotransmission and the immune response (Ahern et al., 2002; Baranano and Snyder, 2001; Bredt and Snyder, 1994). In insects, NO signaling is involved in many physiological processes (Davies, 2000). NO regulates nervous system development and integration (Bicker, 2001; Haase and Bicker, 2003; Schachtner et al., 1999; Seidel and Bicker, 2002; Truman et al., 1996). The hematophagous insect Rhodnius prolixus produces NO, which dilates blood vessels and inhibits platelet aggregation in the host (Ribeiro and Nussenzveig, 1993). Recent studies show that the NO/cGMP pathway is involved in the insect immune response (Imamura et al., 2002; Luckhart et al., 1998; Weiske and Wiesner, 1999) and activation of NO/cGMP signaling inhibits steroid synthesis in the ovary of blow fly (Phormia regina; Maniere et al., 2003). In mammals, there are three NOS genes: neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS) (Bogdan, 2001; Mungrue et al., 2003; Nathan and Xie, 1994). Although their expression and biological roles vary, they share a common structural organization (Ghosh and Salerno, 2003; Kone et al., 2003; Torreilles, 2001). The native enzyme is a homodimer of 130–160-kDa subunits (Torreilles, 2001). The N-terminal oxygenase domain contains the binding motif for a P450-like cysteine thiolate-ligate heme and tetrahydrobiopterin (H4). The C-terminal reductase domain contains the binding motifs for FAD, FMN and NADPH. These two domains are linked by a calmodulin (CaM) binding motif. nNOS and eNOS are constitutively expressed and their enzymatic activities are regulated by the intracellular Ca2+ concentration through binding of Ca2+ to CaM (Roman et al., 2002). They contain a 40–50 amino acid sequence linked to the FMN binding motif that acts as an autoinhibitory loop, blocking electron transfer from FMN to the heme in the absence of Ca2+/CaM (Craig et al., 2002; Ghosh and Salerno, The Journal of Experimental Biology 207, 2845-2857 Published by The Company of Biologists 2004 doi:10.1242/jeb.01117