Spatiotemporal dynamics of guanosine 3',5'-cyclic monophosphate revealed by 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 cGMP indicators by bracketing cGMP-dependent protein kinase (cGPK), minus residues 1-77, between cyan and yellow mutants of green fluorescent protein. cGMP decreased fluorescence resonance energy transfer (FRET) and increased the ratio of cyan to yellow emissions by up to 1.5-fold with apparent dissociation constants of approximately 2 microM and >100:1 selectivity for cGMP over cAMP. To eliminate constitutive kinase activity, Thr(516) of cGPK was mutated to Ala. Emission ratio imaging of the indicators transfected into rat fetal lung fibroblast (RFL)-6 showed cGMP transients resulting from activation of soluble and particulate guanylyl cyclase, respectively, by nitric oxide (NO) and C-type natriuretic peptide (CNP). Whereas all naive cells tested responded to CNP, only 68% responded to NO. Both sets of signals showed large and variable (0.5-4 min) latencies. The phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX) did not elevate cGMP on its own but consistently amplified responses to NO or CNP, suggesting that basal activity 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 parallel fiber activity and NO donors, confirming that single-cell increases in cGMP occur under conditions appropriate to cause synaptic plasticity.

Film-induced sadness as a trigger for disinhibited eating.

OBJECTIVE: We tested whether film-induced sadness enhances food intake in restrained eaters. METHOD: Female participants scoring either high or low on a measure of dietary restraint viewed two film segments in counterbalanced order on successive days: an emotionally neutral travelogue and a sad film depicting the death of the young female protagonist. RESULTS: Contrast analyses revealed that film-induced sadness significantly reduced food intake in low-restraint individuals, but only nonsignificantly increased it in high-restraint individuals. DISCUSSION: When sadness is induced without an apparent ego threat, high-restraint participants may not exhibit as much disinhibited eating as when they are exposed to mood inductions that threaten their self-esteem.