de Wit, C. | bioRxiv Science

bioRxiv2026

Arteriolar GMP signaling revealed by FRET-based real-time measurement in vivo

cGMP evokes arteriolar vasorelaxation and is generated in smooth muscle by the soluble guanylyl cyclase (sGC) stimulated by endothelial NO. Phosphodiesterases (PDEs) degrade cGMP and may contribute in diameter regulation. We examined arteriolar cGMP levels in real-time in vivo upon NO and acetylcholine (ACh) and effects of PDE inhibition. Changes in cGMP were measured by FRET using the indicator protein cGi500. Arterioles in the cremaster muscle of anesthetized mice expressing cGi500 were exposed for imaging. They were stimulated with various NO donors (DEA/NO, SNP, SNAP) and ACh with or without PDE inhibition (non-specific: IBMX; PDE-5 specific: sildenafil). Furthermore, dilations induced by ACh and SNP were studied in sGC-deficient mice. DEA/NO and SNAP led to a fast, concentration-dependent rise of fluorescence ratio of cGi500 (by 4.0% at 10{micro}M), that declined on removal slowly. These ratio changes indicated cGMP increases as verified by inhibition of the sGC using ODQ. PDE inhibition increased cGMP levels slowly despite NO synthase inhibition. However, it did not amplify NO-induced ratio increases, but the decline after drug removal was decelerated. Interestingly, ACh did not modify cGMP levels and dilations were not impaired in sGC-deficient mice. FRET-based imaging allows reliable real-time assessment of arteriolar cGMP levels in vivo. PDE activity does not limit the amplitude, but the duration of cGMP signaling after stimulation. Furthermore, we conclude that ACh does not release endothelial NO in murine arterioles. Preliminary experiments demonstrate that simultaneous measurement of cGMP and diameter is feasible using mice with expression of cGi500 in smooth muscle cells.

physiology↗

bioRxiv2023

The vasodilator-stimulated phosphoprotein (VASP) supports the conduction of vasodilator signals and NO-induced arteriolar dilations in murine arterioles in vivo

VASP is a member of the Enabled/VASP protein family that is involved in cortical actin dynamics and may also contribute to the formation of gap junctions. In vessels, gap junctional coupling allows the transfer of signals along the vessel wall and coordinates vascular behavior. Moreover, VASP is reportedly a mediator of NO-induced inhibition of platelet aggregation. Therefore, we hypothesized that VASP exerts also important physiologic functions in arterioles. We examined the spread of vasodilations enabled by gap junctional coupling in endothelial cells as well as NO-induced arteriolar dilations in VASP-deficient mice by intravital microscopy of the microcirculation in a skeletal muscle in anesthetized mice. Conducted dilations were initiated by brief, locally confined stimulation of the arterioles with acetylcholine. The maximal diameters of the arterioles under study ranged from 30 to 40 m. Brief stimulation with acetylcholine induced a short dilation at the local site that was also observed at remote, upstream sites without an attenuation of the amplitude up to a distance of 1.2 mm in control animals (wild-type). In contrast, remote dilations were reduced in VASP-deficient mice despite a similar local dilation indicating an impairment of conducted dilations. Superfusion of NOdonors induced a concentration-dependent dilation in wild-type mice. However, these dilations were slightly reduced in VASP-deficient animals. In contrast, dilations induced by the endothelial stimulator acetylcholine were fully preserved in VASP-deficient mice. In summary, this study suggests that VASP exerts critical functions in arteriolar diameter control. It is crucial for the conduction of dilator signals along the endothelial cell layer. The impairment possibly reflects a perturbed formation of gap junctions in the endothelial cell membrane. VASP also participates in the full dilatory potential of NOdonors although the effect of its deficiency is only subtle. In contrast, VASP is not required for dilations initiated by endothelial stimulation which are mediated in the murine microcirculation by an EDH-mechanism.

physiology↗