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Sawyer, Carolyn

Pharmacology

2005

PhD

The second messenger guanosine 3', 5'-cyclic monophosphate (cGMP) is implicated in the control and regulation of a yet increasing number of diverse physiological processes. As the appreciation of the importance of understanding the cGMP signaling pathway has grown, so has the awareness of the severely limited availability of techniques with which to study this molecule that appears in rapid intracellular transients. With the increasing acceptance of an important role for cGMP in the mediation of vascular smooth muscle relaxation, indicators were constructed to study the spatial and temporal dynamics of cGMP in living smooth muscle cells. Cygnets, cyclic GMP indicators using energy transfer, were constructed by flanking the conformationally sensitive cGMP receptor cGMP-dependent protein kinase between two fluorescent proteins that participate in fluorescence resonance energy transfer (FRET). Cyclic GMP binds the indicator with an apparent equilibrium dissociation constant of 600nM, causing a conformational change that abolishes FRET between the two fluorophores and providing a means for detecting a change in local cGMP concentrations. In the presence of saturating cGMP, cygnets display a total fluorescence intensity ratio change of 45%. In comparison, cAMP can elicit at most a 10% change in FRET ratio, demonstrating indicator selectivity for the correct cyclic nucleotide. Unlike other methods of cGMP detection, cygnets are genetically encoded, allowing for simple incorporation and use of the indicators in living cells. Furthermore, fluorescent means of detection permits reliable monitoring of highly flexible and reversible intracellular cGMP transients.
Introduced to vascular smooth muscle cells, cygnets demonstrated even cytosolic distribution and nuclear exclusion. FRET changes were measured in response to cGMP accumulation upon activation of particulate or soluble guanylate cyclase with natriuretic peptides or nitric oxide donors, respectively, or by phosphodiesterase inhibition. Cultured rat aortic smooth muscle cells, which exhibit a non-contractile, synthetic phenotype typically seen in response to atherosclerosis or vascular injury, responded to natriuretic peptide-mediated activation of the particulate guanylate cyclase (pGC) and elicited rapid and highly reproducible cGMP transients. Inhibition of either phosphodiesterase 1 or 5 further elevated CNP-induced cGMP levels, suggesting that phosphodiesterases can restrict the cGMP response, limiting cGMP accumulation despite maximal stimulation of the particulate guanylate cyclase. The use of cygnets in human cerebral vascular smooth muscle cells reported the accumulation of cGMP in response to CNP only in the presence ofphosphodiesterase inhibition, suggesting a more stringent control of the cGMP pathway. Furthermore, these cells demonstrated spatial patterns indicative of nonuniform cGMP accumulation in response to particulate guanylate cyclase stimulation and phosphodiesterase inhibition, suggesting the differential localization of cyclases and phosphodiesterases throughout the cell in order to target the cGMP response to specific regions of the cell. In conclusion, the successful construction of FRET-based indicators for the detection of intracellular cGMP has facilitated the temporal resolution and evaluation of the contributions of cyclases and phosphodiesterases in determining overall cGMP accumulation, and the visualization of novel spatial dynamics that will contribute to more fully understanding the role of cGMP in the mediation of smooth muscle relaxation.