The gaseous mediator hydrogen sulfide (H 2 S) is synthesized mainly by cystathionine gammalyase in the heart and plays a role in the regulation of cardiovascular homeostasis. Here we first overview the state of the art in the literature on the cardioprotective effects of H 2 S in various models of cardiac injury. Subsequently, we present original data showing the beneficial effects of parenteral administration of a donor of H 2 S on myocardial and endothelial function during reperfusion in a canine experimental model of cardiopulmonary bypass. Overview of the literature demonstrates that various formulations of H 2 S exert cardioprotective effects in cultured cells, isolated hearts and various rodent and large animal models of regional or global myocardial ischemia and heart failure. In addition, the production of H 2 S plays a role in myocardial pre-and post-conditioning responses. The pathways implicated in the cardioprotective action of H 2 S are multiple and involve K ATP channels, regulation of mitochondrial respiration, and regulation of cytoprotective genes such as Nrf-2. In the experimental part of the current article, we demonstrate the cardioprotective effects of H 2 S in a canine model of cardiopulmonary bypass surgery. Anesthetized dogs were subjected hypothermic cardiopulmonary bypass with 60 minutes of hypothermic cardiac arrest in the presence of either saline (control, n=8), or H 2 S infusion (1 mg/ kg/h for 2 h). Left ventricular hemodynamic variables (via combined pressure-volumeconductance catheter) as well as coronary blood flow, endothelium-dependent vasodilatation to acetylcholine and endothelium-independent vasodilatation to sodium nitroprusside were measured at baseline and after 60 minutes of reperfusion. Ex vivo vascular function and high-energy phosphate contents were also measured. H 2 S led to a significantly better recovery of preload recruitable stroke work (p<0.05) after 60 minutes of reperfusion. Coronary blood flow was also significantly higher in the H 2 S group (p<0.05). While the vasodilatory response to sodium nitroprusside was similar in both groups, acetylcholine resulted in a significantly higher increase in coronary blood flow in the H 2 S-treated group (p<0.05) both in vivo and ex vivo. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript high-energy phosphate contents were better preserved in the H 2 S group. Additionally, the cytoprotective effects of H 2 S were confirmed also using in vitro cell culture experiments in H9c2 cardiac m...

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Acute tubular necrosis is a clinical problem that lacks specific therapy and is characterized by high mortality rate. The ischemic renal injury affects the proximal tubule cells causing dysfunction and cell death after severe hypoperfusion. We utilized a cell-based screening approach in a hypoxia-reoxygenation model of tubular injury to search for cytoprotective action using a library of pharmacologically active compounds. Oxygen-glucose deprivation (OGD) induced ATP depletion, suppressed aerobic and anaerobic metabolism, increased the permeability of the monolayer, caused poly(ADP-ribose) polymerase cleavage and caspase-dependent cell death. The only compound that proved cytoprotective either applied prior to the hypoxia induction or during the reoxygenation was adenosine. The protective effect of adenosine required the coordinated actions of adenosine deaminase and adenosine kinase, but did not requisite the purine receptors. Adenosine and inosine better preserved the cellular ATP content during ischemia than equimolar amount of glucose, and accelerated the restoration of the cellular ATP pool following the OGD. Our results suggest that radical changes occur in the cellular metabolism to respond to the energy demand during and following hypoxia, which include the use of nucleosides as an essential energy source. Thus purine nucleoside supplementation holds promise in the treatment of acute renal failure.

Liver ischemia represents a common clinical problem. In the present study, using an in vitro model of hepatic ischemia-reperfusion injury, we evaluated the potential cytoprotective effect of the purine metabolites, such as adenosine and inosine, and studied the mode of their pharmacological actions. The human hepatocellular carcinoma-derived cell line HepG2 was subjected to combined oxygen-glucose deprivation (COGD; 0-14-24 h), followed by re-oxygenation (0-4-24 h). Adenosine or inosine (300–1,000 μM) were applied in pretreatment. Cell viability and cytotoxicity were measured by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide and lactate dehydrogenase methods, respectively. The results showed that both adenosine and inosine exerted cytoprotective effects, and these effects were not related to receptor-mediated actions, since they were not prevented by selective adenosine receptor antagonists. On the other hand, the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride (EHNA, 10 μM) markedly and almost fully reversed the protective effect of adenosine during COGD, while it did not influence the cytoprotective effect of inosine in the same assay conditions. These results suggest that the cytoprotective effects are related to intracellular actions, and, in the case of adenosine also involve intracellular conversion to inosine. The likely interpretation of these findings is that inosine serves as an alternative source of energy to produce ATP during hypoxic conditions. The protective effects are also partially dependent on adenosine kinase, as the inhibitor 4-amino-5-(3-bromophenyl)-7-(6-morpholino-pyridin-3-yl)pyrido[2,3-d]pyrimidine, 2HCl (ABT 702, 30 μM) significantly reversed the protective effect of both adenosine and inosine during hypoxia and re-oxygenation. Collectively, the current results support the view that during hypoxia, adenosine and inosine exert cytoprotective effects via receptor-independent, intracellular modes of action, which, in part, depend on the restoration of cellular bioenergetics. The present study supports the view that testing of inosine for protection against various forms of warm and cold liver ischemia is relevant.

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This article is part of a themed section on Inventing New Therapies Without Reinventing the Wheel: The Power of Drug Repurposing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.2/issuetoc.

Hypoxia-reoxygenation injury is a commonly used in vitro model of ischemia, which is useful to study the recovery processes following the hypoxic period. Hypoxia can be rapidly induced in vitro by replacing the culture atmosphere with hypoxic or anoxic gas mixture. Cellular injury mostly occurs as a result of energetic failure in this model: the lack of oxygen blocks the mitochondrial respiration and anaerobic metabolism becomes the major source of high-energy molecules in the cells. In the absence of glucose, glycolysis and pentose phosphate pathway fail to suffice the cellular energy prerequisite and longer periods of oxygen-glucose deprivation (OGD) can completely deplete the cellular NAD + and ATP pools. The lack of NAD + results in severe metabolic suppression and predisposes the cells to other injury types. This includes oxidant-induced damage, since oxidative stress activates poly(ADP-ribose) polymerase (PARP) that further depletes the cellular NAD + pool and leads to excessive cell death. The impaired mitochondrial respiration also leads to an increase in the mitochondrial membrane potential and augments the mitochondrial superoxide generation leading to oxidative stress. The above processes ultimately lead to necrotic cell death, but in certain cell types, mitochondrial damage can also trigger apoptosis.

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