Global and local cGMP signals in growth control

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The signalling molecule cGMP controls many cellular functions ranging from growth to contractility and ion transport. The current working model postulates that NO-stimulated guanylyl cyclases generate global cytoplasmic cGMP signals, whereas transmembrane guanylyl cyclases, for instance, the receptors for atrial natriuretic peptide (ANP) or C-type natriuretic peptide (CNP), establish local membrane-associated cGMP microdomains. The physiological outcome of cGMP signal transduction depends on the spatiotemporal profile of the cGMP signal and the prevalence of global vs local cGMP pools. In the present project, we will test this hypothesis by correlative analysis of cGMP signals and cell behaviour in real-time in living cells and tissues of mice, with a focus on the interplay between cGMP and remodelling processes in vascular smooth muscle cells (VSMCs) and dorsal root ganglion (DRG) neurons. In the previous funding period, we have generated transgenic cGMP sensor mice expressing the fluorescence resonance energy transfer (FRET)-based cGi500 sensor either globally in all tissues or selectively in specific cell types. FRET microscopy showed that cGMP signals can indeed be visualized in living primary cells and tissues isolated from cGMP sensor mice. By using a mapping technique, we could directly correlate ANP/CNP responsiveness of individual cultured VSMCs to their respective differentiation state. Interestingly, VSMC populations show pronounced heterogeneity with respect to ANP- and CNP-induced cGMP signals, and the ANP/CNP preference of an individual cell appears to be associated with its growth phenotype. In another subproject, we have established cGMP imaging in primary DRG neurons. We were able to trigger local cGMP signals in subcellular compartments of these neurons, such as axons and growth cones, and to correlate them with Ca(2+) signals. Based on these results, we will now extend our studies to learn more about the mechanisms that shape cGMP signals in VSMCs and DRG neurons, for instance, the role of specific cGMP-degrading phosphodiesterases. We will also analyse the relevance of our findings for vascular remodelling during atherosclerosis and restenosis and for cGMP-mediated axon branching during embryogenesis. Two major questions that will be approached by in vitro and in vivo experiments in mice are (1) how do cGMP signals affect the growth and survival of VSMCs and DRG neurons and (2) how do cell growth and phenotype affect cGMP signalling in these cell types? To achieve our goals, we will closely interact with the other groups of this research unit to share expertise as well as mouse and cell models. The visualization of cGMP during signal transduction in a living mammalian cell or organism will be useful for both basic research on cyclic nucleotides as well as for target identification and drug development.