• Top
• Oral presentation

Oral presentation

Recently, we developed genetically encoded cGMP-indicators (Honda et al., (2001), Proc. Natl. Acad. Sci. USA, 98, 2437-2442) and employed them to study the spatial and temporal dynamics of intracellular cGMP in single living vascular smooth muscle cells. We have demonstrated that fluorescence resonance energy transfer (FRET) provides a valuable methodological basis for the development of such indicators, which we named Cygnets: cyclic GMP indicators using energy transfer. Our Cygnets have enabled us to probe the elusive intracellular cGMP transients in response to physiological and pharmacological stimuli in a variety of cell types in unprecedented resolution and detail. A central issue in vascular biology is the signaling pathways by which cGMP mediates nitric oxide (NO) induced vasodilatation. Progress in this area has been severely hampered by the lack of specific cGMP indicators, which allow to measure transient cGMP signals in live smooth muscle cells fast, efficient and reliable. Cygnets present a unique solution to this problem by uniting the powers of molecular biology, protein biochemistry and fluorescence microscopy imaging. We now report the expression of our most recent indicator, Cygnet-2.1 and the ability to monitor spatial and temporal changes of cGMP levels in primary smooth muscle cells derived from rat aorta, human mesenteric, omental and cerebral arteries. All smooth muscle cells showed robust and highly reproducible cGMP transients in response to C-type natriuretic peptide (CNP). However, nitric oxide donors elicited cell type specific cGMP transients of highly characteristic kinetics. Our recent results also suggest that cyclic nucleotide phosphodiesterases play a key role in maintaining and modulating intracellular cGMP levels. In addition, we could recently show that upon stimulation, a subset of all cell types tested so far, display peculiar spatially unidirectional wave patterns of intracellular cGMP. In summary, our cygnets have provided fundamental new information about the factors that regulate intracellular cGMP and their kinetic and three-dimensional relationships in vascular smooth muscle.

Have something to say? Post a comment on this article!