whereas others reported immunocytochemical evidence for astrocytic eNOS (see the recent review of the literature by Lin et al., 2007). Tests with eNOS-knockout mice would help determine whether the astrocyte staining is speciﬁc or not. In the meantime, endothelial cells are probably the major, if not the sole, location of eNOS in brain tissue. Endothelial eNOS is of emerging relevance in the regulation of brain function independently of its role in the vasculature (see below) and it continues to be the subject of much research, not least because of its importance in cardiovascular function and malfunction. The controls over eNOS activity are multifarious (reviewed in Cirino et al., 2003; Dudzinski et al., 2006). Catalytically active eNOS in endothelial cells is largely tied to the plasma membrane by a lipid modiﬁcation (palmitoylation) and resides in specialised invaginations (caveoli) in association with other proteins, including caveolin-1 and heat-shock protein (Hsp)-90. Dissociation from caveolin-1 is required for activity and is promoted by Ca2+ ⁄ calmodulin binding. Despite this, eNOS is often tonically active in blood vessels, perhaps the most important mechanism for sustaining the activity being phosphorylation on serine-1179, enabling the enzyme to function at resting cytosolic Ca2+ concentrations. The prototype kinase mediating serine-1179 phosphorylation is Akt, which is activated by phosphoinositide-3 kinase in response to stimuli such as shear stress, oestrogens, insulin and vascular endothelial growth factor, but the same site is also targeted by AMP kinase (in response to metabolic stress), protein kinase C, cAMP- and cGMP-dependent protein kinases, and CaMKII. NO receptors The activation by NO of ‘soluble’ guanylyl cyclase in homogenates of various tissues was discovered well in advance of any inkling that NO was of biological relevance (Arnold et al., 1977; Miki et al., 1977), and proved vital to the hypothesis that the endothelium-derived relaxing factor was NO (Furchgott, 1999; Ignarro, 1999). It remains the only recognized physiological NO signal transduction mechanism and much evidence has accrued during the last several years conﬁrming its pre-eminence in transducing the actions of endogenous NO (Krumen- acker et al., 2004), the most striking example of which being the complete loss of NO-mediated vascular relaxation following genetic deletion of NO-activated guanylyl cyclases (Friebe et al., 2007). The homogenate-based name (soluble guanylyl cyclase) is still widely used but it has little meaning in a cellular context, and was never meant to (Chrisman et al., 1975). In reality, the proteins are enzyme-linked receptors that, in cells, are often associated indirectly with membranes (see below) and here they are simply called ‘NO receptors’. In common with all other receptors, NO receptors are equipped with a ligand binding site and a transduction domain but in many ways they are especially fascinating. The ligand binding site is an unremarkable haem group of the type used in haemoglobin for binding O2 but, when incorporated into the receptor protein, it exhibits amazing preference for NO, allowing cellular NO signalling to occur in the presence of > 10 000-fold excess of O2, despite the close chemical similarity of the two ligands (Stone & Marletta, 1994; Martin et al., 2006). NO detection by protein-associated haems appears to have evolved in certain bacteria and to have been incorporated into eukaryotic animals by horizontal gene transfer (Iyer et al., 2003; Fitzpatrick et al., 2006). The protein component is an ab-heterodimer of which there are two known isoforms, a1b1 and a2b1. They comprise a haem-binding region, a dimerisation domain and a catalytic domain where GTP is converted to cGMP. The catalytic domain is very similar to that of adenylyl cyclase, to the extent that switching three amino acids converts NO-activated guanylyl cyclase into NO-activated adenylyl Neural nitric oxide transmission 2785 cyclase (Sunahara et al., 1998). In the inactive state, the ligand- binding haem group is coordinated to the protein by a histidine bond (Wedel et al., 1994; Zhao et al., 1998) and, by analogy with adenylyl cyclase, the catalytic domain in the inactive state is in an open conﬁguration (Dessauer et al., 1999). Binding of NO to the haem is almost diffusion-limited (Makino et al., 1999; Zhao et al., 1999) and, from structural studies on a homologous cyanobacterial NO sensor (Ma et al., 2007), this event causes the haem to pivot which, together with a dislocation of the coordinating histidine group, results in a conformational change in the protein that propagates to the catalytic domain, resulting in domain closure and catalysis. When studied in a cell-free environment, the a1b1 and a2b1 NO receptor isoforms are similarly sensitive to NO, half-maximal activity being evoked at a concentration of 1 nm (Wykes & Garthwaite, 2004). Other properties, such as the maximal guanylyl cyclase activity and pharmacological properties, also appear very similar (Russwurm et al., 1998; Gibb et al., 2003) but whether this pertains to the proteins in their natural environment is not known. The potency of NO for its receptors in cells (EC50 = 10 nm) is an order of magnitude lower than that of the puriﬁed protein (Mo et al., 2004; Roy & Garthwaite, 2006), which is partly explained by the presence in cells of ATP (which, although inhibitory, accelerates the kinetics) and a lower GTP concentration than is typically used in biochemical assays (B. Roy, E.J. Halvey and J. Garthwaite, unpublished observations). In brain cells, NO switches on the associated guanylyl cyclase activity with no observable delay (with a 20-ms sampling time) and, on removal of NO, the activity decays with a half-time of 200 ms (Bellamy & Garthwaite, 2001b), kinetics not dissimilar to that of NMDA receptors or of metabotropic GABA or glutamate receptors (Dale & Roberts, 1985; Dutar & Nicoll, 1988; Batchelor & Garthwaite, 1997). Although widespread, the two NO receptor isoforms have a differing cellular distribution in the brain (Budworth et al., 1999; Gibb & Garthwaite, 2001; Mergia et al., 2003; Szabadits et al., 2007) and, at the subcellular level, may also have different locations because the a2b1 receptor binds through its PDZ domain to proteins that are enriched in synapses, namely PSD-95 and putatively also to the related proteins PSD-93, SAP-97 and SAP-102 (Russwurm et al., 2001). The other isoform, a1b1, may be cytosolic in part but there is evidence that it too may become plasma membrane-associated under conditions of raised intracellular Ca2+ (Zabel et al., 2002) or exposure to canna- binoids (Jones et al., 2008). Membrane association may depend on binding to Hsp-90 (Venema et al., 2003; Papapetropoulos et al., 2005; Nedvetsky et al., 2008), Hsp-70 (Balashova et al., 2005) or other proteins (Meurer et al., 2004). In homogenates, varying proportions of total NO-activated guanylyl cyclase activity are found in membrane fractions (Arnold et al., 1977): for example, in rat platelets, this component amounts to 60% of the activity in the cytosol whereas, in the cerebellum, it is half this amount (Wykes & Garthwaite, 2004). In homogenates of platelets and cerebellum, NO was equipotent (EC50 1 nm) on the receptors in supernatant and membranes (Wykes & Garthwaite, 2004). The biological signiﬁcance of the shuttling of a1b1 NO receptors to the membrane (and, presumably, back again) is unknown. The linkage between a2b1 receptors and membrane- associated synaptic proteins may position them within close range of the sites of NO release, which would be particularly important if NO signalling operates in discrete spatial dimensions (see below). Conceivably, by placing the receptor closer to an NO source, attachment of the a1b1 isoform to the outer cell membrane could perform a similar function. In subcellular dimensions, this positioning could make the difference between the receptors being accessible to NO generated in a nearby cellular compartment and being out of range (see below). ª The Author (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd European Journal of Neuroscience, 27, 2783–2802