Selected compounds (original paper) Comments ODQ (Garthwaite et al., 1995) and NS 2028 (Olesen et al., 1998) YC-1 (Ko et al., 1994) and BAY 41-2272 (Stasch et al., 2001) Protoporphyrin IX (Ignarro et al., 1982), BAY 58-2667 (Stasch et al., 2002) and HMR1766, S3448 (Schindler et al., 2006) 2786 J. Garthwaite Table 2. Pharmacology of NO receptors Class of agent Inhibitors of ligand binding Allosteric enhancers Ligand binding site (haem) mimetics As judged by immunocytochemistry of NO receptor proteins (Ding et al., 2004) and of cGMP following exposure to NO sources (Southam & Garthwaite, 1993; de Vente et al., 1998), NO receptors are present to a greater or lesser extent throughout the CNS and have a distribution complementary to that of nNOS, consistent with the two being functional partners. A growing number of good pharmacological tools targeting different sites are now available for manipulating NO– cGMP signalling at the receptor level (Table 2). NO receptors are potential substrates for phosphorylation by several kinases but physiological regulation through such modiﬁcations remains to be clariﬁed (reviewed in Pyriochou & Papapetropoulos, 2005). Recent evidence indicates that phosphorylation by cAMP-dependent protein kinase enhances the associated guanylyl cyclase activity at resting levels of NO in pituitary cells (Kostic et al., 2004) whereas, in gastrointestinal smooth muscle cells, activation of muscarinic M2 receptors reduces cGMP generation through Src kinase-dependent tyrosine phosphorylation of the receptor (Murthy, 2008). Transduction of cGMP signals Direct actions of cGMP can be exerted by binding to agonist or regu- latory sites on cyclic nucleotide-gated (CNG) ion channels (reviewed in Kaupp & Seifert, 2002) or hyperpolarization-activated, cyclic nucleo- tide-modulated (HCN) channels (reviewed in Craven & Zagotta, 2006). cGMP also binds directly to the phosphodiesterase (PDE) enzymes PDE2, PDE5 and, in retinal photoreceptor cells, PDE6, resulting in heightened catalytic activity and cGMP breakdown. cGMP is a low- efﬁcacy substrate for another PDE, PDE3, so that cGMP binding to the catalytic site leads to inhibition of cAMP hydrolysis, potentially raising cAMP levels (reviewed by Bender & Beavo, 2006). Probably the most widespread mechanism employed by cGMP is activation of PKG, which exists in three forms, PKG1a and PKG1b (splice variants) and PKGII, which is anchored to the plasma membrane by myristoylation. PKG1a is concentrated in cerebellum and dorsal root ganglia and PKG1b in the hippocampus and olfactory bulb, whereas PKGII has a more widespread distribution in the brain but shows a particular abundance in the thalamus (reviewed in Feil et al., 2005; Vaandrager et al., 2005; Hofmann et al., 2006). Several substrates for PKG have been identiﬁed (reviewed in Schlossmann & Hofmann, 2005) and many of its actions are exerted at the level of phosphatases, leading indirectly to increased or decreased levels of phosphorylation of effector proteins. Whilst traditionally acting intracellularly, cGMP is also found extracellularly in the brain, where its levels ﬂuctuate according to Act by oxidising the haem iron, thereby inhibiting NO binding (Schrammel et al.,1996); oxidation may predispose the receptors to haem loss and then protein degradation (Stasch et al., 2006). Potentiate NO-evoked activity by slowing the rate of deactivation (Friebe & Koesling, 1998). YC-1 also affects cGMP hydrolysing phosphodiesterases at similar concentrations to those active on the receptor (Galle et al., 1999), whereas BAY 41-2272 is more selective (but see Bischoff & Stasch, 2004; Mullershausen et al., 2004). Activate the haem-free species (Schmidt et al., 2004; Roy et al., 2008) which normally appears to be a very small proportion of the total but which can nevertheless evoke functional responses when engaged pharmacologically. Protoporphyrin IX is a partial agonist and zinc protoporphyrin IX an antagonist for this site; the haem-free receptor targeted by the compounds may become more abundant in disease states (Stasch et al., 2006). changes in endogenous NO formation (reviewed by Vincent et al., 1998; Pepicelli et al., 2004). cGMP can be exported from cells through members of the multidrug resistance protein family (reviewed by Sager, 2004) and might serve an additional intercellular signalling role, consistent with evidence that extracellularly applied cGMP has biological effects (Touyz et al., 1997; Poulopoulou & Nowak, 1998; Montoliu et al., 1999). Hydrolysis of cGMP Most of the 11 known PDE families can hydrolyse cGMP, those with the highest afﬁnity being PDE1, 2, 3, 5, 6, 9, 10 and 11 (see review by Bender & Beavo, 2006). Little is known about the PDE isoforms responsible for cGMP hydrolysis in individual cell types, and there may be mixtures at work. For example, cerebellar Purkinje cells all appear to contain PDE5 but a subset additionally expresses a PDE1 isoform (Shimizu-Albergine et al., 2003); in cerebellar astrocytes PDE5 is also prominent but PDE4, which has low afﬁnity (> 100 lm) for cGMP ( 1 lm for cAMP), also contributes when cGMP reaches high levels (Bellamy & Garthwaite, 2001a); in ventrobasal thalamic neurones, on the other hand, PDE2 and putatively PDE9 appear most important (Hepp et al., 2007); PDE2 also plays a major role in hydrolyzing NO-evoked increases in cGMP levels in hippocampus and striatum (van Staveren et al., 2001; Wykes et al., 2002; Boess et al., 2004) whereas, in pituitary nerve terminals, PDE5 is again signiﬁcant (Zhang et al., 2007b). Suitably selective inhibitors, now available for a number of PDEs, give a complementary strategy for investigating NO receptor signalling at various levels, including in memory performance in vivo (reviewed by Blokland et al., 2006). Other transduction pathways There are some examples of physiological NO signals being transduced in a cGMP-independent manner in the nervous system (Jacoby et al., 2001; Lev-Ram et al., 2002), implying the existence of other NO receptors yet to be identiﬁed. One chemical process that has been hailed by some as a physiological NO signal transducing mechanism is the nitrosation of protein thiol groups (see Stamler et al., 2001), or S-nitrosation (often confusingly called S-nitrosylation; see discussion by Koppenol, 2002), but this idea remains highly controversial. It is facile to evoke protein S-nitrosation by exposing cells to high concentrations of nitrosothiols (e.g. S-nitroso-N-acetylpenicillamine) which can transfer their NO ª The Author (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd European Journal of Neuroscience, 27, 2783–2802