A primer for measuring cGMP signaling and cGMP-mediated vascular relaxation

Abstract

Soluble guanylyl cyclase (sGC, also called GC1) is the main receptor for nitric oxide (NO) that catalyzes the production of the second messenger molecule, 3'5' cyclic guanosine monophosphate (cGMP) leading to vasorelaxation, and inhibition of leukocyte recruitment and platelet aggregation. Enhancing cGMP levels, through sGC agonism or inhibition of cGMP breakdown via phosphodiesterase inhibition, has yielded FDA approval for several cGMP modifier therapies for treatment of cardiovascular and pulmonary diseases. While basic research continues to improve our understanding of cGMP signaling and as new therapies evolve to elevate cGMP levels, we provide a short methodological primer for measuring cGMP and cGMP-mediated vascular relaxation for investigators.

Keywords: Catalytic activity; Guanylyl cyclase; Mesenteric arteries; Myography; Nitric oxide; VASP; cGMP.

Fig. 1.

Setup for cGMP separation.

Fig. 2.. Overview of thoracic aorta and mesenteric vasculature anatomy.

a) Schematic of a mouse showing anatomic location of a mouse thoracic aorta and mesentery arteries. b) Image of a mouse thoracic aorta with heart shown in upper left corner. c) Zoomed in image of anatomic location of mesenteric artery cascade. d) Schematic showing first, second and third order mesenteric arteries. e) Image of a pinned out mesenteric. f) Zoomed image of a mesentery vein (left vessel, dark red) and an artery (right, light pink and white).

Fig. 3.. Overview of aorta and mesenteric artery myography setup and standard protocol for assessing cGMP-mediated vascular relaxation.

A) Image of a 4-chamber DMT620 myograph used to assess aortic and mesenteric artery function. B) Image of a single 2-pin setup for an aorta or C) wire setup for a mesenteric artery. Images below show mounted aorta and mesenteric artery. D) Schematic diagram illustrating the NO-sGC-cGMP pathway and agonists used to determine NO-dependent and NO-independent vasodilation. E) A schematic workflow of a myography experiment assessing vasodilatation to sGC modulator compounds.