Endogenous hydrogen sulfide (H 2 S) is involved in the regulation of vascular tone. We hypothesized that the lowering of calcium and opening of potassium (K) channels as well as calciumindependent mechanisms are involved in H 2 S-induced relaxation in rat mesenteric small arteries. Amperometric recordings revealed that free [H 2 S] after addition to closed tubes of sodium hydrosulfide (NaHS), Na 2 S, and GYY4137 [P-(4-methoxyphenyl)-P-4-morpholinyl-phosphinodithioic acid] were, respectively, 14%, 17%, and 1% of added amount. The compounds caused equipotent relaxations in isometric myographs, but based on the measured free [H 2 S], GYY4137 caused more relaxation in relation to released free H 2 S than NaHS and Na 2 S in rat mesenteric small arteries. Simultaneous measurements of [H 2 S] and tension showed that 15 mM of free H 2 S caused 61% relaxation in superior mesenteric arteries. Simultaneous measurements of smooth muscle calcium and tension revealed that NaHS lowered calcium and caused relaxation of NE-contracted arteries, while high extracellular potassium reduced NaHS relaxation without corresponding calcium changes. In NE-contracted arteries, NaHS (1 mM) lowered the phosphorylation of myosin light chain, while phosphorylation of myosin phosphatase target subunit 1 remained unchanged. Protein kinase A and G, inhibitors of guanylate cyclase, failed to reduce NaHS relaxation, whereas blockers of voltage-gated K V 7 channels inhibited NaHS relaxation, and blockers of mitochondrial complex I and III abolished NaHS relaxation. Our findings suggest that low micromolar concentrations of free H 2 S open K channels followed by lowering of smooth muscle calcium, and by another mechanism involving mitochondrial complex I and III leads to uncoupling of force, and hence vasodilation.
Nitric
oxide (NO) is a highly potent but short-lived endogenous radical with
a wide spectrum of physiological activities. In this work, we developed
an enzymatic approach to the site-specific synthesis of NO mediated
by biocatalytic surface coatings. Multilayered polyelectrolyte films
were optimized as host compartments for the immobilized β-galactosidase
(β-Gal) enzyme through a screen of eight polycations and eight
polyanions. The lead composition was used to achieve localized production
of NO through the addition of β-Gal–NONOate, a prodrug
that releases NO following enzymatic bioconversion. The resulting
coatings afforded physiologically relevant flux of NO matching that
of the healthy human endothelium. The antiproliferative effect due
to the synthesized NO in cell culture was site-specific: within a
multiwell dish with freely shared media and nutrients, a 10-fold inhibition
of cell growth was achieved on top of the biocatalytic coatings compared
to the immediately adjacent enzyme-free microwells. The physiological
effect of NO produced via the enzyme prodrug therapy was validated
ex vivo in isolated arteries through the measurement of vasodilation.
Biocatalytic coatings were deposited on wires produced using alloys
used in clinical practice and successfully mediated a NONOate concentration-dependent
vasodilation in the small arteries of rats. The results of this study
present an exciting opportunity to manufacture implantable biomaterials
with physiological responses controlled to the desired level for personalized
treatment.
Nitric oxide (NO) is a potent biological molecule that contributes to a wide spectrum of physiological processes. However, the full potential of NO as therapeutic agents is significantly complicated by its short half-life and limited diffusion distance in human tissues. Current strategies for NO delivery focus on encapsulation of NO donors into prefabricated scaffolds or an enzyme-prodrug therapy approach. The former is limited by the finite pool of NO donors available, while the latter is challenged by the inherent low stability of natural enzymes. This work provides the first report of zinc oxide (ZnO) particles with innate glutathione peroxidase and glycosidase activities, a combination that allows to catalytically decompose both endogenous (S-nitrosoglutathione) and exogenous (β-gal-NONOate) donors to generate NO at physiological conditions. By tuning the concentration of ZnO particles and NO prodrugs, physiologically relevant NO levels are achieved. ZnO preserves its catalytic property for at least 6 months and the activity of ZnO in generating NO from prodrugs in human serum is demonstrated. The ZnO catalytic activity will be beneficial towards generating stable NO release for long-term biomedical applications.
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