Spatiotemporal dynamics of guanosine 3 ',5 '-cyclic monophosphate revealedby a genetically encoded, fluorescent indicator

To investigate the dynamics of guanosine 3',5'-cyclic monophosphate (cGMP) in single living cells, we constructed genetically encoded, fluorescent cGM P indicators by bracketing cGMP-dependent protein kinase (cGPK), minus resi dues 1-77, between cyan and yellow mutants of green fluorescent protein, cG MP decreased fluorescence resonance energy transfer (FRET) and increased th e ratio of cyan to yellow emissions by up to 1.5-fold with apparent dissoci ation constants of approximate to2 muM and >100:1 selectivity for cGMP over cAMP. To eliminate constitutive kinase activity, Thr(516) of cGPK was muta ted to Ala. Emission ratio imaging of the indicators transfected into rat f etal lung fibroblast (RFL)-6 showed cGMP transients resulting from activati on of soluble and particulate guanylyl cyclase, respectively, by nitric oxi de (NO) and C-type natriuretic peptide (CNP). Whereas all naive cells teste d responded to CNP, only 68% responded to NO. Both sets of signals showed l arge and variable (0.5-4 min) latencies. The phosphodiesterase (PDE) inhibi tor 3-isobutyl-1-methylxanthine (IBMX) did not elevate cGMP on its own but consistently amplified responses to NO or CNP, suggesting that basal activi ty of guanylate cyclase is very low and emphasizing the importance of PDEs in cGMP recycling. A fraction of RFL cells showed slowly propagating tides of cGMP spreading across the cell in response to delocalized application of NO. Biolistically transfected Purkinje neurons showed cGMP responses to pa rallel fiber activity and NO donors, confirming that single-cell increases in cGMP occur under conditions appropriate to cause synaptic plasticity.