FRET-based real-time measurement of cGMP in skeletal muscle arterioles in vivo

Endothelial NO regulates arteriolar tone by activating sGC, which produces cGMP evoking vasorelaxation. Phosphodiesterases (PDE) degrade cGMP and may limit resulting cGMP increases. In this study, we examined cGMP levels in arterioles in vivo during NO application and PDE inhibition. Transgenic mice expressing a cGMP indicator protein were prepared for intravital microscopy. Arterioles (diameter: ~50µm) in the cremaster muscle were exposed for FRET-based cGMP imaging by removing perivascular tissue. The muscle was sequentially superfused with acetylcholine, adenosine and NO donors with or without PDE inhibitors (IBMX, sildenafil) and cGMP levels continuously monitored. DEA and SNAP led to fast, concentration-dependent rises of the CFP/YFP ratio (from 0.3 to 4.0% change; at 0.1 to 10µM) indicative of increases in intracellular cGMP that declined slowly on removal. In contrast, SNP did not alter the ratio. Adenosine, an activator of adenylyl cyclase, did not enhance the ratio, verifying specificity. Surprisingly, acetylcholine did not modify the ratio during intact eNOS. IBMX and sildenafil increased the ratio (by 3.5%) despite NOS inhibition. In their presence, SNAP-induced ratio changes were not enhanced, however, the decline upon SNAP removal was slowed. Changes of intracellular cGMP can be determined in real-time in vivo. Exogenous NO and PDE inhibition enhance cGMP levels even in the absence of eNOS activity. Since PDE inhibition only slows the decline of enhanced ratios upon removal of NO, cGMP degradation is not the limiting factor for cGMP increase during sGC stimulation. Furthermore, we conclude that acetylcholine does not activate eNOS in the murine microcirculation.