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.
Highlights
Nitric oxide (NO) is produced in small puffs by the nitric oxide synthase of endothelial cells, which diffuses into vascular smooth muscle (VSM) and acts directly on the NO-sensitive guanylyl cyclases α1β1/α2β1 (Arnold et al, 1977; Moncada et al, 1991a; Lowenstein and Michel, 2006)
Through the unique coupling of clamped NO delivery to small pressurized arteries and FlincG-transfected single VSM cells, we report here that the driving force behind vasomotor reactivity is both the dynamic behavior of NO, as well as its exquisitely high sensitivity toward sGC in the vasculature
As cGMP is a critical mediator for the regulation of vascular relaxation, and blood flow and blood pressure, understanding how [cGMP]i is managed in VSM cells provides invaluable information to the molecular mechanisms underlying vascular physiology
Summary
Nitric oxide (NO) is produced in small puffs by the nitric oxide synthase of endothelial cells (eNOS), which diffuses into vascular smooth muscle (VSM) and acts directly on the NO-sensitive guanylyl cyclases α1β1/α2β1 (Arnold et al, 1977; Moncada et al, 1991a; Lowenstein and Michel, 2006). It has been recently shown that the coupling of NO donors to the NO-scavenger CPTIO alters the kinetics of NO as to deliver a sustained application of NO, which can be modeled to determine NO concentration, previously confirmed by NO electrode (Bellamy et al, 2002; Griffiths et al, 2003). This approach allowed for the direct vascular response to NO to be determined without interference of NO decay. VSM cells appear to harbor their unique sets of regulatory enzymes in the maintenance of cGMP homeostasis
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