Nitric oxide (NO) functions as a diffusible transmitter in most tissues of the body and exerts its effects by binding to receptors harboring a guanylyl cyclase transduction domain, resulting in cGMP accumulation in target cells. Despite its widespread importance, very little is known about how this signaling pathway operates at physiological NO concentrations and in real time. To address these deficiencies, we have exploited the properties of a novel cGMP biosensor, named δ-FlincG, expressed in cells containing varying mixtures of NO-activated guanylyl cyclase and cGMPhydrolyzing phosphodiesterase activity. Responsiveness to NO, signifying a physiologically relevant rise in cGMP to 30 nM or more, was seen at concentrations as low as 1 pM, making cells by far the most sensitive NO detectors yet encountered. Even cells coexpressing phosphodiesterase-5, a cGMP-activated isoform found in many NO target cells, responded to NO in concentrations as low as 10 pM. The dynamics of NO capture and signal transduction was revealed by administering timed puffs of NO from a local pipette. A puff lasting only 100 ms, giving a calculated peak intracellular NO concentration of 23 pM, was detectable. The results could be encapsulated in a quantitative model of cellular NO-cGMP signaling, which recapitulates the NO responsiveness reported previously from crude cGMP measurements on native cells, and which explains how NO is able to exert physiological effects at extremely low concentrations, when only a tiny proportion of its receptors would be occupied.is an evolutionarily ancient transmitter of fundamental importance to the physiology of the mammalian cardiovascular, nervous, and other systems (1, 2). NO signal transduction takes place through the simplest one-component type of receptor, comprising an NO binding site (a prosthetic heme) coupled to a guanylyl cyclase (GC) transduction domain (3, 4). The elevation in cellular cGMP that follows NO binding is curtailed by one or more phosphodiesterase (PDE) enzymes that convert it to GMP. There have been countless descriptions of changes in the levels of cGMP in association with NOmediated transmission in different cells and tissues. It has become increasingly evident, however, that the levels of cGMP measurable by traditional methods, such as radioimmunoassay, may not be relevant to physiological NO signaling. For example, smooth muscle relaxation (5) and cGMP-dependent phosphorylation events in platelets (6) are seen at NO concentrations below those giving increases in cGMP measurable with such methods; NO can still relax vascular smooth muscle despite deletion of 94% of the NO-activated GC (7); and engagement of 2% or less of the available GC activity is sufficient to stimulate cGMPdependent phosphorylation in platelets (8). These findings indicate that cells possess a very large receptor excess and that new approaches are required to understand the elementary properties of NO capture and signal amplification by cells under physiological conditions. A related gap in knowledge is in...

The upregulation of eNOS in the LyECs of CH rats causes long-term lymphatic remodelling, which is characterised by a loss of SMC lymphatic coverage. The amelioration of this lymphatic abnormality by chronic eNOS inhibition results in improved lymphatic drainage and reduced ascites.

Objective Palmitoylation, the reversible addition of the lipid palmitate to a cysteine, can alter protein localization, stability, and function. The ZDHHC family of protein acyl transferases catalyzes palmitoylation of numerous proteins. The role of ZDHHC enzymes in intact tissue and in vivo is largely unknown. Herein, we characterize vascular functions in a mouse that expresses a nonfunctional ZDHHC21 (“F233Δ”). Approach and Results Physiological studies of isolated aortae and mesenteric arteries from F233Δ mice revealed an unexpected defect in responsiveness to phenylephrine, an α1 adrenergic receptor agonist. In vivo, F233Δ mice displayed a blunted response to infusion of phenylephrine and were found to have elevated catecholamine levels and elevated vascular α1 adrenergic receptor gene expression. Telemetry studies showed that the F233Δ mice were tachycardic and hypotensive at baseline, consistent with diminished vascular tone. In biochemical studies, ZDHHC21 was shown to palmitoylate the α1D adrenoceptor, and to interact with it in a molecular complex, thus suggesting a possible molecular mechanism by which the receptor can be regulated by ZDHHC21. Conclusions Together the data support a model in which ZDHHC21 F233Δ diminishes the function of vascular α1 adrenergic receptors, leading to reduced vascular tone which manifests in vivo as hypotension and tachycardia. This is to our knowledge the first demonstration of a ZDHHC isoform affecting vascular function in vivo and identifies a novel molecular mode of regulation of vascular tone and blood pressure.

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.

Real-time and noninvasive imaging of intracellular second messengers in mammalian cells, while preserving their in vivo phenotype, requires biosensors of exquisite constitution. Here we provide the methodology for utilizing the single wavelength cGMP-biosensor δ-FlincG in aortic vascular smooth muscle cells.