Zinc Oxide Particles Catalytically Generate Nitric Oxide from Endogenous and Exogenous Prodrugs.

Nitric oxide (NO) is a potent biological molecule that contributes to a wide spectrum of physiological processes. However, the full potential of NO as a therapeutic agent 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. Zinc oxide (ZnO) particles with innate glutathione peroxidase and glycosidase activities, a combination that allows to catalytically decompose both endogenous (S-nitrosoglutathione) and exogenous (ß-gal-NONOate) donors to generate NO at physiological conditions are reported. 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 toward generating stable NO release for long-term biomedical applications.

Simultaneous Measurements of Tension and Free H2S in Mesenteric Arteries.

Hydrogen sulfide (H2S), in addition to nitric oxide and carbon monoxide, is the third gasotransmitter and known to cause relaxation in peripheral arteries. Here we describe a method that allows simultaneous measurement of contractility in arteries mounted in an isometric wire myograph and the concentration of free H2S in the lumen of the artery as well as in the organ bath. This method can be used to directly correlate how much free H2S is needed to cause relaxation, which previously has been difficult to answer as H2S can be found in many different forms.

Extracellular l-arginine Enhances Relaxations Induced by Opening of Calcium-Activated SKCa Channels in Porcine Retinal Arteriole.

We investigated whether the substrate for nitric oxide (NO) production, extracellular l-arginine, contributes to relaxations induced by activating small (SKCa) conductance Ca2+-activated potassium channels. In endothelial cells, acetylcholine increased 3H-l-arginine uptake, while blocking the SKCa and the intermediate (IKCa) conductance Ca2+-activated potassium channels reduced l-arginine uptake. A blocker of the y+ transporter system, l-lysine also blocked 3H-l-arginine uptake. Immunostaining showed co-localization of endothelial NO synthase (eNOS), SKCa3, and the cationic amino acid transporter (CAT-1) protein of the y+ transporter system in the endothelium. An opener of SKCa channels, cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (CyPPA) induced large currents in endothelial cells, and concentration-dependently relaxed porcine retinal arterioles. In the presence of l-arginine, concentration-response curves for CyPPA were leftward shifted, an effect unaltered in the presence of low sodium, but blocked by l-lysine in the retinal arterioles. Our findings suggest that SKCa channel activity regulates l-arginine uptake through the y+ transporter system, and we propose that in vasculature affected by endothelial dysfunction, l-arginine administration requires the targeting of additional mechanisms such as SKCa channels to restore endothelium-dependent vasodilatation.

Identification and Directed Development of Non-Organic Catalysts with Apparent Pan-Enzymatic Mimicry into Nanozymes for Efficient Prodrug Conversion.

Nanozymes, nanoparticles that mimic the natural activity of enzymes, are intriguing academically and are important in the context of the Origin of Life. However, current nanozymes offer mimicry of a narrow range of mammalian enzymes, near-exclusively performing redox reactions. We present an unexpected discovery of non-proteinaceous enzymes based on metals, metal oxides, 1D/2D-materials, and non-metallic nanomaterials. The specific novelty of these findings lies in the identification of nanozymes with apparent mimicry of diverse mammalian enzymes, including unique pan-glycosidases. Further novelty lies in the identification of the substrate scope for the lead candidates, specifically in the context of bioconversion of glucuronides, that is, human metabolites and privileged prodrugs in the field of enzyme-prodrug therapies. Lastly, nanozymes are employed for conversion of glucuronide prodrugs into marketed anti-inflammatory and antibacterial agents, as well as "nanozyme prodrug therapy" to mediate antibacterial measures.

Enzyme Prodrug Therapy Achieves Site-Specific, Personalized Physiological Responses to the Locally Produced Nitric Oxide.

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 ß-galactosidase (ß-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 ß-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.

Involvement of Potassium Channels and Calcium-Independent Mechanisms in Hydrogen Sulfide-Induced Relaxation of Rat Mesenteric Small Arteries.

Endogenous hydrogen sulfide (H2S) is involved in the regulation of vascular tone. We hypothesized that the lowering of calcium and opening of potassium (K) channels as well as calcium-independent mechanisms are involved in H2S-induced relaxation in rat mesenteric small arteries. Amperometric recordings revealed that free [H2S] after addition to closed tubes of sodium hydrosulfide (NaHS), Na2S, 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 [H2S], GYY4137 caused more relaxation in relation to released free H2S than NaHS and Na2S in rat mesenteric small arteries. Simultaneous measurements of [H2S] and tension showed that 15 µM of free H2S 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 KV7 channels inhibited NaHS relaxation, and blockers of mitochondrial complex I and III abolished NaHS relaxation. Our findings suggest that low micromolar concentrations of free H2S 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.

Involvement of hydrogen sulfide in perivascular and hypoxia-induced inhibition of endothelin contraction in porcine retinal arterioles.

Perivascular retina has been shown to regulate retinal vascular tone. In the present study, we evaluated an ex vivo retina preparation, and investigated whether hydrogen sulfide (H2S) mediates an inhibitory effect of retina and/or hypoxia on arteriolar tone. In retina, immunolabeling showed an increase of glial fibrillary acidic protein, but not vimentin over time in Müller cells, and the presence of necrotic cells after 2 h and apoptotic cells after 8 h. Isometric tension recordings showed endothelin-1(ET-1) to induce concentration-dependent contractions, which were reduced in the presence of retina. In arterioles with retina no change was observed in ET-1 contractions after 5 h compared to 8 h. Hypoxia (1% O2) reduced ET-1 contraction in arterioles with and without retina. The H2S donor, GYY4137 and the salt, sodium hydrogen sulfide, induced concentration-dependent relaxations in ET-1 contracted retinal arterioles. Inhibition of the H2S producing enzymes, cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE), with carboxymethoxylamine (AOA) and L-propargylglycine (PPG) enhanced ET-1 contractions. This effect was more pronounced in hypoxic conditions. However, even in the presence of AOA and PPG ET-1 induced less contraction in the presence of perivascular retina compared to isolated vessels. These findings suggest that both the presence of perivascular retina and hypoxia reduce arteriolar vasoconstriction and that both H2S and another factor mediate this effect. Finally, H2S donors, as well as endogenous H2S, can reduce retinal arteriolar tone, suggesting a potential therapeutic role for enhanced H2S bioavailability in the treatment of retinal disease.