Phosphodiesterase type 2 and the homeostasis of cyclic GMP in living thalamic neurons R. Hepp, L. Tricoire, E. Hu, N. Gervasi, D. Paupardin-Tritsch, B. Lambolez and P. Vincent Universite ´ Pierre et Marie Curie-Paris6, CNRS, UMR 7102, Paris, France Abstract The ubiquitous second messenger cyclic GMP (cGMP) is synthesized by soluble guanylate cyclases in response to nitric oxide (NO) and degraded by phosphodiesterases (PDE). We studied the homeostasis of cGMP in living thalamic neurons by using the genetically encoded fluorescence resonance energy transfer sensor Cygnet, expressed in brain slices through viral gene transfer. Natriuretic peptides had no effect on cGMP. Basal cGMP levels decreased upon inhibition of NO synthases or soluble guanylate cyclases and increased when PDEs were inhibited. Single cell RT-PCR analysis showed that thalamic neurons express PDE1, PDE2, PDE9, and PDE10. Basal cGMP levels were increased by the PDE2 inhibitors erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) and BAY60-7550 but were unaffected by PDE1 or PDE10 inhibitors. We conclude that PDE2 regulates the basal cGMP concentration in thalamic neurons. In addition, in the presence of 3-isobutyl-1-methylxanthine (IBMX), cGMP still decreased after application of a NO donor. Probenecid, a blocker of cGMP transporters, had no effect on this decrease, leaving PDE9 as a possible candidate for decreasing cGMP concentration. Basal cGMP level is poised at an intermediate level from which it can be up or down-regulated according to the cyclase and PDE activities. Keywords: fluorescence resonance energy transfer imaging, cyclic GMP, homeostasis, nitric oxide, phosphodiesterases, single-cell RT-PCR. J. Neurochem. (2007) 102, 1875–1886. The cyclic GMP (cGMP) signaling pathway has been linked to numerous important functions in the central nervous system, including neurotransmitter release (Bredt and Snyder 1994), the modulation of neuronal excitability, and synaptic plasticity processes such as long-term potentiation and long-term depression (Lev-Ram et al. 1997; Daniel et al. 1998; Monfort et al. 2004; Feil et al. 2005). cGMP is produced by soluble guanylate cyclases (sGC) following their activation by nitric oxide (NO), or by membrane- associated guanylate cyclases activated upon binding of natriuretic peptides. cGMP is degraded by phosphodiest- erases (PDE) or extruded from the cells by transporters (Borst et al. 2000). To date, 11 families of PDEs have been identified, based on their structure, substrate specificity, and pharmacological profile (Beavo 1995; Conti and Jin 1999; Lugnier 2006). Among PDEs that hydrolyze cGMP, only PDE1, PDE2, PDE5, PDE9, and PDE10 are expressed in the brain (Beavo 1995; Menniti et al. 2006). PDE9 degrades solely cGMP with a K m of 70–170 nmol/L (Fisher et al. 1998; Huai et al. 2004). In contrast PDE1, 2, 5, and 10 hydrolyse both cyclic AMP (cAMP) and cGMP with a K m for cGMP around 1–10 lmol/L. PDE1 are calcium/calmodulin activated, PDE2 and PDE5 are stimulated by cGMP and PDE10 is stimulated by cAMP (Gross-Langenhoff et al. 2006). The main target of cGMP is the cGMP-dependent protein kinases (PKG), which is widely expressed in the CNS (El-Husseini et al. 1999). In addition, cGMP directly activates cyclic nucleotide gated channels, but the functional presence of these channels in central neurons is still unclear (Kaupp and Seifert 2002). Received December 2, 2006; revised manuscript received March 23, 2007; accepted April 5, 2007. Address correspondence and reprint requests to R. Hepp, Univer- site ´ Pierre et Marie Curie-Paris 6, CNRS, UMR 7102, 9 quai Saint Bernard, F-75005 Paris, France. E-mail: regine.hepp@snv.jussieu.fr Abbreviations used: cAMP, cyclic AMP; CFP, cyan fluorescent pro- tein; cGMP, cyclic GMP; DMSO, dimethyl sulfoxide; DEANO, 2-(N, N- diethylamino)-diazenolate-2-Oxide; EHNA, erythro-9-(2-hydroxy-3-no- nyl) adenine; FRET, fluorescence resonance energy transfer; IBMX, 3- isobutyl-1-methylxanthine; L-NNA, N-x-nitro-L-arginine; NO, nitric oxide; NOS, nitric oxide synthase; ODQ, 1H-[1,2,4]oxadiazole[4,3- a]quinoxalin-1-one; PBS, phosphate buffer saline; PDE, phosphodiest- erase; PKG, cGMP-dependent protein kinases; scPCR, single cell RT- PCR; sGC, soluble guanylate cyclases; SNAP, S-nitroso-n-acetylpenic- illamine; VB, ventro basal complex of the thalamus; YFP, yellow fluorescent protein. Journal of Neurochemistry , 2007, 102, 1875–1886 doi:10.1111/j.1471-4159.2007.04657.x Ó 2007 The Authors Journal Compilation Ó 2007 International Society for Neurochemistry, J. Neurochem. (2007) 102, 1875–1886 1875