Sub-Nanomolar Sensitivity of Nitric Oxide Mediated Regulation of cGMP and Vasomotor Reactivity in Vascular Smooth Muscle

Abstract

Nitric oxide (NO) is a potent dilator of vascular smooth muscle (VSM) by modulating intracellular cGMP ([cGMP]i) through the binding and activation of receptor guanylyl cylases (sGC). The kinetic relationship of NO and sGC, as well as the subsequent regulation of [cGMP]i and its effects on blood vessel vasodilation, is largely unknown. In isolated VSM cells exposed to both pulsed and clamped NO we observed transient and sustained increases in [cGMP]i, with sub-nanomolar sensitivity to NO (EC50 = 0.28 nM). Through the use of pharmacological inhibitors of sGC, PDE5, and PKG, a comprehensive VSM-specific modeling algorithm was constructed to elucidate the concerted activity profiles of sGC, PDE5, phosphorylated PDE5, and PDE1 in the maintenance of [cGMP]i. In small pressure-constricted arteries of the resistance vasculature we again observed both transient and sustained relaxations upon delivery of pulsed and clamped NO, while maintaining a similarly high sensitivity to NO (EC50 = 0.42 nM). Our results propose an intricate dependency of the messengers and enzymes involved in cGMP homeostasis, and vasodilation in VSM. Particularly, the high sensitivity of sGC to NO in primary tissue indicates how small changes in the concentrations of NO, irrespective of the form of NO delivery, can have significant effects on the dynamic regulation of vascular tone.

Traumatic Brain Injury Disrupts Cerebrovascular Tone Through Endothelial Inducible Nitric Oxide Synthase Expression and Nitric Oxide Gain of Function

“…Furthermore, NO electrodes lack the spatial resolution to determine the vascular component of TBI‐induced increases in NO. Our present study used a unique combination of live‐NO imaging using the diaminofluorescein NO indicator DAF‐2 DA 57 and an NO‐clamp approach 11,38 that delivers set concentrations of NO to examine NO levels in isolated cerebral arteries 24 hours after fluid percussion–induced TBI in rats.…”

Real-Time Monitoring the Spatiotemporal Dynamics of Intracellular cGMP in Vascular Smooth Muscle Cells

“…PDE5 is unique in that it can be phosphorylated to enhance and prolong its activity [3, 19–25]. The level of phosphorylation has been shown to be essential for cGMP dynamics in VSM cells [26], and less critical in platelets, astrocytes, and sGC-transfected HEK cells [27–29]. This differential in the level of involvement in regulating [cGMP] i seems to be cell- and tissue-type specific.…”

“…FlincGs include fragments of the PKGIα regulatory domain containing the two cGMP-binding domains fused to cpEGFP on the N-terminus [42]. Particularly, the variant δ-FlincG exhibited high cGMP specificity, rapid binding, and dissociation kinetics and the capacity for confocal imaging, providing the first evidence for distinct spatial localization of cGMP signals in VSM cells [26, 42]. δ-FlincG has provided vertical progress by granting the most accurate method for cGMP detection applicable to many different cell types [26, 28, 42–47].…”

“…Particularly, the variant δ-FlincG exhibited high cGMP specificity, rapid binding, and dissociation kinetics and the capacity for confocal imaging, providing the first evidence for distinct spatial localization of cGMP signals in VSM cells [26, 42]. δ-FlincG has provided vertical progress by granting the most accurate method for cGMP detection applicable to many different cell types [26, 28, 42–47]. FlincG-type biosensors have helped to overcome the two major roadblocks in determining VSM signaling: (1) smooth muscle cells lose the expression of many smooth muscle-specific markers with culturing [38], and (2) nitric oxide has an extremely short biological half-life [48–50], which creates an experimental dilemma whereby small signaling events may be missed with traditional immunoblotting or kinase assays.…”

“…Using FlincGs have allowed us to elicit the separate localization of the cytosolic NO- and membrane-restricted natriuretic peptide (NP)-induced cGMP in VSM cells, as well as create cells capable of detecting picomolar concentrations of cGMP produced by sGC [28, 42]. We have also determined the temporal kinetic relationship of NO and NO-induced cGMP, and which PDEs are critical in its maintenance in VSM [26]. …”