Neuroprotective Actions of Hydrogen Sulfide-Releasing Compounds in Isolated Bovine Retinae.

Background: We have evidence that hydrogen sulfide (H2S)-releasing compounds can reduce intraocular pressure in normotensive and glaucomatous rabbits by increasing the aqueous humor (AH) outflow through the trabecular meshwork. Since H2S has been reported to possess neuroprotective actions, the prevention of retinal ganglion cell loss is an important strategy in the pharmacotherapy of glaucoma. Consequently, the present study aimed to investigate the neuroprotective actions of H2S-releasing compounds against hydrogen peroxide (H2O2)-induced oxidative stress in an isolated bovine retina. Materials and Methods: The isolated neural retinae were pretreated with a substrate for H2S biosynthesis called L-cysteine, with the fast H2S-releasing compound sodium hydrosulfide, and with a mitochondrial-targeting H2S-releasing compound, AP123, for thirty minutes before a 30-min oxidative insult with H2O2 (100 µM). Lipid peroxidation was assessed via an enzyme immunoassay by measuring the stable oxidative stress marker, 8-epi PGF2α (8-isoprostane), levels in the retinal tissues. To determine the role of endogenous H2S, studies were performed using the following biosynthesis enzyme inhibitors: aminooxyacetic acid (AOAA, 30 µM); a cystathione-ß-synthase/cystathionine-γ-lyase (CBS/CSE) inhibitor, α-ketobutyric acid (KBA, 1 mM); and a 3-mercaptopyruvate-s-sulfurtransferase (3-MST) inhibitor, in the absence and presence of H2S-releasing compounds. Results: Exposure of the isolated retinas to H2O2 produced a time-dependent (10-40 min) and concentration-dependent (30-300 µM) increase in the 8-isoprostane levels when compared to the untreated tissues. L-cysteine (10 nM-1 µM) and NaHS (30 -100 µM) significantly (p < 0.001; n = 12) prevented H2O2-induced oxidative damage in a concentration-dependent manner. Furthermore, AP123 (100 nM-1 µM) attenuated oxidative H2O2 damage resulted in an approximated 60% reduction in 8-isoprostane levels compared to the tissues treated with H2O2 alone. While AOAA (30 µM) and KBA (1 mM) did not affect the L-cysteine evoked attenuation of H2O2-induced oxidative stress, KBA reversed the antioxidant responses caused by AP123. Conclusions: In conclusion, various forms of H2S-releasing compounds and the substrate, L-cysteine, can prevent H2O2-induced lipid peroxidation in an isolated bovine retina.

Glutathione Modulates Hydrogen Sulfide Release and the Ocular Hypotensive Action of Diallyl Polysulfide Compounds.

BACKGROUND: Hydrogen sulfide (H2S) is an endogenous transmitter with the potential to regulate aqueous humor dynamics and protect retinal neurons from degeneration. The aim of the present study was two-fold: (a) to evaluate the release of H2S from two polysulfides, diallyl disulfide (DADS), and diallyl trisulfide (DATS); and (b) to investigate their ocular hypotensive actions in normotensive male and female rabbits in the presence and absence of GSH. MATERIALS AND METHODS: H2S was quantified hourly for up to 6 h using a H2S-Biosensor (World Precision Instruments, Sarasota, Fl). Intraocular pressure (IOP) was assessed in normotensive New Zealand Albino rabbits using a pneumotonometer (model 30 classic; Reichert Ophthalmic Instruments, Depew, NY, USA). RESULTS: In the presence of GSH, there was an increase in the in vitro release of H2S produced by DADS and DATS. Both DADS and DATS also caused a dose-dependent reduction in IOP in male and female rabbits, in both treated and untreated eyes. For instance, in male animals, the presence of GSH (3% and 5%) significantly (p < 0.05, n = 5) enhanced the ocular hypotensive action of DADS (2%) and DATS (2%) from 14.02 ± 2.89% to 18.67 ± 5.6% and from 16.22 ± 3.48 to 23.62 ± 5.79%, respectively. CONCLUSIONS: GSH enhanced both H2S release and ocular hypotensive action of the polysulfides in a manner that was dependent on the number of sulfur atoms present in each polysulfide. Furthermore, female animals were less sensitive to the IOP-lowering action of the polysulfides, when compared to their male counterparts.

Correction: Heruye et al. Current Trends in the Pharmacotherapy of Cataracts. Pharmaceuticals 2020, 13, 15.

In the original publication [...].

Tolerability, safety, and immunogenicity of the novel oral polio vaccine type 2 in children aged 6 weeks to 59 months in an outbreak response campaign in The Gambia: an observational cohort study.

BACKGROUND: Novel oral polio vaccine type 2 (nOPV2) has been used to interrupt circulating vaccine-derived poliovirus type 2 outbreaks following its WHO emergency use listing. This study reports data on the safety and immunogenicity of nOPV2 over two rounds of a campaign in The Gambia. METHODS: This observational cohort study collected baseline symptoms (vomiting, diarrhoea, irritability, reduced feeding, and reduced activity) and axillary temperature from children aged 6 weeks to 59 months in The Gambia before a series of two rounds of a nOPV2 campaign that took place on Nov 20-26, 2021, and March 19-22, 2022. Serum and stool samples were collected from a subset of the participants. The same symptoms were re-assessed during the week following each dose of nOPV2. Stool samples were collected on days 7 and 28, and serum was collected on day 28 following each dose. Adverse events, including adverse events of special interest, were documented for 28 days after each campaign round. Serum neutralising antibodies were measured by microneutralisation assay, and stool poliovirus excretion was measured by real-time RT-PCR. FINDINGS: Of the 5635 children eligible for the study, 5504 (97·7%) received at least one dose of nOPV2. There was no increase in axillary temperature or in any of the baseline symptoms following either rounds of the campaigns. There were no adverse events of special interest and no other safety signals of concern. Poliovirus type 2 seroconversion rates were 70% (95% CI 62 to 78; 87 of 124 children) following one dose of nOPV2 and 91% (85 to 95; 113 of 124 children) following two doses. Poliovirus excretion on day 7 was lower after the second round (162 of 459 samples; 35·3%, 95% CI 31·1 to 39·8) than after the first round (292 of 658 samples; 44·4%, 40·6 to 48·2) of the campaign (difference -9·1%; 95% CI -14·8 to -3·3), showing the induction of mucosal immunity. INTERPRETATION: In a campaign in west Africa, nOPV2 was well tolerated and safe. High rates of seroconversion and evidence of mucosal immunity support the licensure and WHO prequalification of this vaccine. FUNDING: Bill & Melinda Gates Foundation.

Epidemiology of measles cases, vaccine effectiveness, and performance towards measles elimination in The Gambia.

INTRODUCTION: In 2011, member states of the World Health Organization (WHO) Africa Regional Office (AFRO) resolved to eliminate Measles by 2020. Our study aims to assess The Gambia's progress towards the set AFRO measles elimination target and highlight surveillance and immunisation gaps to better inform future measles prevention strategies. MATERIAL AND METHODS: A retrospective review of measles surveillance data for the period 2011-2019, was extracted from The Gambia case-based measles surveillance database. WHO-UNICEF national coverage estimates were used for estimating national level MCV coverage. Measles post campaign coverage survey coverage estimates were used to estimate national measles campaign coverage. RESULTS: One hundred and twenty-five of the 863 reported suspected cases were laboratory confirmed as measles cases. More than half (53.6%) of the confirmed cases have unknown vaccination status, 24% of cases were vaccinated, 52.8% of cases occurred among males, and 72.8% cases were among urban residents. The incidence of measles cases per million population was lowest (0) in 2011-2012 and highest in 2015 and 2016 (31 and 23 respectively). The indicator for surveillance sensitivity was met in all years except in 2016 and 2019. Children aged 5-9 years (Incidence Rate Ratio-IRR = 0.6) and residents of Central River region (IRR = 0.21) had lower measles risk whilst unvaccinated (Adjusted IRR = 5.95) and those with unknown vaccination status (IRR 2.21) had higher measles risk. Vaccine effectiveness was 89.5%. CONCLUSION: The Gambia's quest to attain measles elimination status by 2020 has registered significant success but it is unlikely that all target indicators will be met. Vaccination has been very effective in preventing cases. There is variation in measles risk by health region, and it will be important to take it into account when designing prevention and control strategies. The quality of case investigations should be improved to enhance the quality of surveillance for decision making.

Current Trends in the Pharmacotherapy of Cataracts.

Cataracts, one of the leading causes of preventable blindness worldwide, refers to lens degradation that is characterized by clouding, with consequent blurry vision. As life expectancies improve, the number of people affected with cataracts is predicted to increase worldwide, especially in low-income nations with limited access to surgery. Although cataract surgery is considered safe, it is associated with some complications such as retinal detachment, warranting a search for cheap, pharmacological alternatives to the management of this ocular disease. The lens is richly endowed with a complex system of non-enzymatic and enzymatic antioxidants which scavenge reactive oxygen species to preserve lens proteins. Depletion and/or failure in this primary antioxidant defense system contributes to the damage observed in lenticular molecules and their repair mechanisms, ultimately causing cataracts. Several attempts have been made to counteract experimentally induced cataract using in vitro, ex vivo, and in vivo techniques. The majority of the anti-cataract compounds tested, including plant extracts and naturally-occurring compounds, lies in their antioxidant and/or free radical scavenging and/or anti-inflammatory propensity. In addition to providing an overview of the pathophysiology of cataracts, this review focuses on the role of various categories of natural and synthetic compounds on experimentally-induced cataracts.

Standardization of a new method for assessing the development of cataract in cultured bovine lenses.

PURPOSE: To standardize a new method for assessing cataractogenesis in isolated cultured bovine lenses using L-cysteine as the standard anti-cataract agent. METHODS: Intact bovine lenses were cultured in DMEM with L-cysteine in presence or absence of hydrogen peroxide (H2O2). Lens opacity (transmittance) was determined using a plate reader. Lens homogenate glutathione (GSH) and superoxide dismutase (SOD) contents were measured using enzyme immunoassays kits. RESULTS: DMEM-cultured lenses exhibited a time-dependent loss in transmittance (230-710 nm) up to 120 h, achieving the highest reduction of 38.6 ±â€¯0.09% at 420 nm (p < .001;n = 12). Compared to untreated lenses (time in hours [t] = 0), L-cysteine (10-6 M and 10-5 M) significantly (p < .001;n = 6) increased time-dependent transmittance (420 nm) by 31.6 ±â€¯0.17% and 28.0 ±â€¯0.07%(t = 120), respectively. When compared to DMEM-cultured lenses (t = 0), H2O2 (10 mM, 50 mM and 100 mM) significantly (p < .001;n = 12) reduced transmittance by 57.8 ±â€¯0.1, 57.4 ±â€¯0.04 and 87.7 ±â€¯0.6%(t = 120), respectively. Moreover, L-cysteine significantly (p < .001;n = 6) attenuated H2O2 (50 mM)-induced decrease in transmittance by 12.5 ±â€¯0.05%(10-6 M), 13.0 ±â€¯0.09%(10-5 M), 14.5 ±â€¯0.08%(10-4 M) and 8.6 ±â€¯0.11%(10-3 M)(t = 120), respectively. When compared to untreated lenses (t = 0), the time-dependent decrease (p < .001;n = 5) in lenticular total GSH content and total SOD activity of 46.1 ±â€¯0.06% and 42.0 ±â€¯1.65% (t = 120) was attenuated (p < .001;n = 5) by L-cysteine (10-6 M) by 76.6 ±â€¯0.06% and 7.4 ±â€¯1.98%, respectively. Similarly, the H2O2(50 mM)-induced decline (p < .001; n = 5) in total GSH content and SOD activity of 82.6 ±â€¯0.08% and 86.6 ±â€¯0.66% (t = 120) was attenuated by L-cysteine (10-4 M) by 74.7 ±â€¯1.05% and 161.1 ±â€¯4.9%, respectively. CONCLUSION: Measurement of spectral transmission coupled with assessment of the activity of antioxidant enzymes in bovine cultured lens can provide a useful tool in studies of cataracts in an animal model of this disease.

Serotonin-2B/2C Receptors Mediate Bovine Ciliary Muscle Contraction: Role in Intraocular Pressure Regulation.

PURPOSE: To study the pharmacological profile of the serotonin (5-hydroxytryptamine [5-HT]) receptor subtype mediating contractions in bovine isolated ciliary muscles. METHODS: Ciliary muscle strips were isolated from bovine eyeballs and mounted in organ baths containing aerated (95% O2, 5% CO2) Krebs buffer solution maintained at 37°C. Each muscle strip was attached at 1 end to a Grass Force-displacement Transducer connected to a Polyview Computer System for recording changes in isometric tension. After an equilibration period, ciliary muscle strips were exposed to selective agonists and antagonists of 5-HT receptors. RESULTS: Both selective and nonselective agonists for 5-HT produced concentration-dependent contractions of isolated ciliary muscles with the following rank order of potency: BW723C86>α-methyl-5-HT>MK-212>>8-hydroxy-DPAT>quipazine>R-DOI>>5-HT>>tryptamine. The selective 5-HT2 receptor antagonists, M-100907 (5-HT2A), RS-127445 (5-HT2B), and RS-102221 (5-HT2C), produced noncompetitive inhibition of the contractile effects of selective agonists yielding antagonist potency (pKB) values of 251 ± 27.2 nM (n = 4), 52.5 ± 6.3 nM (n = 4), and 79.4 ± 9.5 nM (n = 4), respectively. CONCLUSION: On the basis of the profile of activity of selective agonists and antagonists, we conclude that the 5-HT2B and 5-HT2C receptor subtypes appear to be the predominant serotonin receptors that mediate the contractile action of this amine in bovine isolated ciliary muscles.

Comparative Effects of Hydrogen Sulfide-Releasing Compounds on [3H]D-Aspartate Release from Bovine Isolated Retinae.

We investigated the pharmacological actions of a slow-releasing H2S donor, GYY 4137; a substrate for the biosynthesis of H2S, L-cysteine and its precursor, N-acetylcysteine on potassium (K+; 50 mM)-evoked [3H]D-aspartate release from bovine isolated retinae using the Superfusion Method. GYY 4137 (10 nM-10 µM), L-cysteine (100 nM-10 µM) and N-acetylcysteine (10 µM-1 mM) elicited a concentration-dependent decrease in K+-evoked [3H]D-aspartate release from isolated bovine retinae without affecting basal tritium efflux. At equimolar concentration of 10 µM, the rank order of activity was as follows: L-cysteine > GYY 4137 > N-acetylcysteine. A dual inhibitor of the biosynthetic enzymes for H2S, cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE), amino-oxyacetic acid (AOA; 3 mM) reversed the inhibitory responses caused by GYY 4137, L-cysteine and N-acetylcysteine on K+-evoked [3H]D-aspartate release. Glibenclamide (300 µM), an inhibitor of KATP channels blocked the inhibitory action of GYY 4137 and L-cysteine but not that elicited by N-acetylcysteine on K+-induced [3H]D-aspartate release. The inhibitory effect of GYY 4137 and L-cysteine on K+-evoked [3H]D-aspartate release was reversed by the non-specific inhibitor of nitric oxide synthase (NOS), L-NAME (300 µM). Furthermore, a specific inhibitor of inducible NOS (iNOS), aminoguanidine (10 µM) blocked the inhibitory action of L-cysteine on K+-evoked [3H]D-aspartate release. We conclude that both donors and substrates for H2S production can inhibit amino acid neurotransmission in bovine isolated retinae, an effect that is dependent, at least in part, upon the intramural biosynthesis of this gas, and on the activity of KATP channels and NO synthase.

Pharmacology of Serotonin Receptors Causing Contraction of Isolated Bovine Posterior Ciliary Arteries: Role in Ocular Blood Flow.

PURPOSE: To determine the serotonergic (5HT) receptor subtype mediating the contraction of bovine posterior ciliary arteries (BPCAs) in vitro. METHODS: Longitudinal isometric tension was measured in BPCA strips (4-5 mm) mounted in 25 mL organ baths containing oxygenated Krebs solution at 37°C. Cumulative contractile concentration-response (C-R) curves were generated for various 5-HT agonists to assess their potencies and maximal degrees of contraction. Multiple agonist C-R curves were also constructed in the presence and absence of receptor-selective antagonists to determine antagonist potencies using Schild plots. RESULTS: Selective and nonselective agonists for 5-HT receptors elicited concentration-dependent contractile responses in BPCAs with the following rank order of potency: MK-212 > BW723C86 > α-methyl-5-HT >5-methoxy-α-5-methyl-5-HT >> R-DO1 > >5-HT >> cabergoline >> 5-methoxy-dimethyl-tryptamine >> 2-methyl-5-HT >> tryptamine. Interestingly, both 8-OH-DPAT (5HT1A agonist) and quipazine (5HT3 agonist) did not elicit contractions in BPCAs. The contractions produced by BW723C86 (5-HT2B agonist) were antagonized by 5-HT receptor blockers, RS-127445 (5-HT2B antagonist), and M-100907 (5-HT2A antagonist), yielding antagonist pA2 values of 7.5 ± 0.12 (n = 4) and 6.2 ± 0.17 (n = 4), respectively. Furthermore, contractions elicited by MK-212 (5-HT2C agonist) was blocked by RS-102221 (5-HT2C antagonist), although noncompetitively. CONCLUSIONS: On the basis of the pharmacological profile of selective agonists and antagonists, we conclude that serotonin-induced contractions of the BPCA are mediated primarily by a combination of 5HT2C and/or 5HT2B receptors. It appears that 5-HT1A and 5-HT3 receptors are not involved in the contractile action of BPCAs to serotonin.

Regulation of Aqueous Humor Dynamics by Hydrogen Sulfide: Potential Role in Glaucoma Pharmacotherapy.

Hydrogen sulfide (H2S) is a gaseous transmitter with well-known biological actions in a wide variety of tissues and organs. The potential involvement of this gas in physiological and pathological processes in the eye has led to several in vitro, ex vivo, and in vivo studies to understand its pharmacological role in some mammalian species. Evidence from literature demonstrates that 4 enzymes responsible for the biosynthesis of this gas (cystathionine ß-synthase, CBS; cystathionine γ-lyase, CSE; 3-mercaptopyruvate sulfurtransferase, 3MST; and d-amino acid oxidase) are present in the cornea, iris, ciliary body, lens, and retina. Studies of the pharmacological actions of H2S (using several compounds as fast- and slow-releasing gas donors) on anterior uveal tissues reveal an effect on sympathetic neurotransmission and the ability of the gas to relax precontracted iris and ocular vascular smooth muscles, responses that were blocked by inhibitors of CSE, CBS, and KATP channels. In the retina, there is evidence that H2S can inhibit excitatory amino acid neurotransmission and can also protect this tissue from a wide variety of insults. Furthermore, exogenous application of H2S-releasing compounds was reported to increase aqueous humor outflow facility in an ex vivo model of the porcine ocular anterior segment and lowered intraocular pressure (IOP) in both normotensive and glaucomatous rabbits. Taken together, the finding that H2S-releasing compounds can lower IOP and can serve a neuroprotective role in the retina suggests that H2S prodrugs could be used as tools or therapeutic agents in diseases such as glaucoma.

Regulation of Excitatory Amino Acid Transmission in the Retina: Studies on Neuroprotection.

Excitotoxicity occurs in neurons due to the accumulation of excitatory amino acids such as glutamate in the synaptic and extrasynaptic locations. In the retina, excessive glutamate concentrations trigger a neurotoxic cascade involving several mechanisms, including the elevation of intracellular calcium (Ca2+) and the activation of α-amino-3-hydroxy 5-methyl-4-iso-xazole-propionic acid/kainate (AMPA/KA) and N-methyl-d-aspartate (NMDA) receptors leading to retinal degeneration. Both ionotropic glutamate receptors (iGluRs) and metabotropic glutamate receptors (mGluRs) are present in the mammalian retina. Indeed, due to the abundant expression of GluRs, the mammalian retina is highly susceptible to excitotoxic neurodegeneration. Excitotoxicity has been postulated to present a common downstream mechanism for several stimuli, including hypoglycemia, hypoxia, ischemia, and chronic neurodegenerative diseases. Experimental approaches to the study of neuroprotection in the retina have utilized insults that trigger hypoxia, hypoglycemia, or excitotoxicity. Using these experimental approaches, the neuroprotective potential of GluR agents, including the NMDA receptor modulators (MK801, ifenprodil, memantine); AMPA/KA receptor antagonist (CNQX); Group II and III mGluR agonists (LY354740, quisqualate); and Ca2+-channel blockers (diltiazem, lomerizine, verapamil, ω-conotoxin), and others (pituitary adenylate cyclase activating polypeptide, neuropeptide Y, acetylcholine receptor agonists) have been elucidated. In addition to corroborating the exocytotic role of excitatory amino acids in retinal degeneration, these studies affirm that multiple mechanism/s contribute to the prevention of damage caused by excitotoxicity in the retina. Therefore, it is feasible that several pathways are involved in protecting the retina from toxic insults in ocular neurodegenerative conditions such as glaucoma and retinal ischemia. Furthermore, these experimental models are viable tools for evaluating therapeutic candidates in ocular neuropathies.

Effects of Hydrogen Sulfide-Releasing Compounds on Aqueous Humor Outflow Facility in Porcine Ocular Anterior Segments, Ex Vivo.

PURPOSE: To investigate the pharmacological actions of hydrogen sulfide (H2S)-releasing compounds l-cysteine and sodium hydrosulfide (NaHS) on aqueous humor (AH) outflow facility in porcine ocular anterior segment. METHODS: Porcine ocular anterior segments were perfused with Dulbecco's modified Eagle's medium at a constant pressure of 7.35 mmHg. After stable outflow baseline, explants were exposed to NaHS or l-cysteine. The increase in outflow generated by the H2S-releasing compounds was measured in the absence and presence of inhibitor of H2S biosynthesis (aminooxyacetic acid; AOAA), blocker of KATP channels (glibenclamide), and inhibitor of adenylyl cyclase (SQ 22536). Hematoxylin and eosin (H&E) staining was used to assess trabecular meshwork (TM) morphology. RESULTS: l-cysteine elicited a concentration-dependent increase in AH outflow facility, reaching maximal effect at 100 nM (150.6% ± 17.2% of basal level). This increase in outflow induced by l-cysteine was significantly (P < 0.001) antagonized by AOAA (30 µM) and glibenclamide (100 µM). AOAA and glibenclamide had no significant action on baseline outflow, whereas SQ 22536 (100 µM) increased outflow for only an hour. In addition, NaHS produced a concentration-dependent increase in AH outflow, with a maximal effect at 10 µM (151.4% ± 22.9% of basal level). Likewise, the increase in outflow caused by NaHS was significantly (P < 0.04) blocked by glibenclamide and SQ 22536. H&E staining revealed that l-cysteine or NaHS did not alter TM conformation. CONCLUSION: H2S-releasing compounds can increase outflow facility in porcine ocular anterior segment. The stimulatory action of these compounds on outflow is mediated, in part by endogenously produced H2S, KATP channels, and adenylyl cyclase.

Effect of Hydrogen Sulfide Donors on Intraocular Pressure in Rabbits.

PURPOSE: In this study, we investigated the effect of a slow-releasing hydrogen sulfide (H2S) donor, GYY 4137, on intraocular pressure (IOP) in normotensive rabbits. Furthermore, we compared the IOP-lowering action of GYY 4137 with those elicited by other H2S-producing compounds, l-cysteine and ACS67 (a hybrid compound of latanoprost with an H2S-releasing moiety). METHODS: IOP was measured in New Zealand normotensive male albino rabbits using a pneumatonometer (model 30 classic; Reichert Ophthalmic Instruments, Depew, NY). At 0 h, 50 µL of test compounds were applied topically to 1 eye of each animal, while the contralateral eye received the same quantity of vehicle (saline). IOP was measured hourly until baseline IOP readings were attained and animal eyes monitored for potential side effects (i.e., tearing, hyperemia). RESULTS: GYY 4137 (0.1%-2%) produced a dose-dependent decrease in IOP reaching a maximum of 27.8% ± 3.14% (n = 5) after 6 h. Interestingly, a significant contralateral effect was observed in vehicle-treated controls eyes at all doses tested. l-cysteine (5%) and ACS67 (0.005%) also elicited a significant (P < 0.01) decrease in IOP that achieved a maximum of 28.84% ± 1.53% (n = 5) and 23.27% ± 0.51% (n = 5), respectively, after 3 h. All 3 H2S-producing compounds also caused a significant contralateral effect in vehicle-treated control eyes. CONCLUSION: We conclude that GYY 4137 and other H2S-producing donors can reduce IOP in normotensive rabbits. However, the profile of IOP-lowering action of GYY 4137 was different from the other H2S donors affirming its ability to act as a slow-releasing gas donor.

Pharmacological Actions of Hydrogen Sulfide Donors on Sympathetic Neurotransmission in the Bovine Anterior Uvea, In Vitro.

In the present study, we investigated the effect of three different sources of hydrogen sulfide (H2S) on sympathetic neurotransmission from isolated superfused bovine iris-ciliary bodies. The three agents under consideration were: ACS67, a hybrid of latanoprost and a H2S-donating moiety; L-cysteine, a substrate for endogenous production of H2S and GYY 4137, a slow donor of H2S. We also examined the contribution of prostaglandins to the pharmacological actions of the H2S donors on release of [(3)H]-norepinephrine ([(3)H]NE) triggered by electrical field stimulation. ACS67, L-cysteine and GYY 4137 caused a concentration-dependent inhibition of electrically-evoked [(3)H]NE release from isolated bovine iris-ciliary bodies without affecting basal [(3)H]NE efflux. The cyclooxygenase inhibitor, flurbiprofen enhanced the inhibitory action of ACS67 and L-cysteine on stimulated [(3)H]NE release. Both aminooxyacetic acid, an inhibitor of cystathionine-ß-synthase and glibenclamide, a KATP channel blocker reversed the inhibition of evoked NE release induced by the H2S donors. We conclude that H2S donors can inhibit sympathetic neurotransmission from isolated bovine iris-ciliary bodies, an effect partially dependent on the in situ production of H2S and prostanoids, and is mediated by an action on KATP channels.

Inhibitory action of novel hydrogen sulfide donors on bovine isolated posterior ciliary arteries.

In the present study, we investigate the inhibitory effect of novel H2S donors, AP67 and AP72 on isolated bovine posterior ciliary arteries (PCAs) under conditions of tone induced by an adrenoceptor agonist. Furthermore, we examined the possible mechanisms underlying the AP67- and AP72-induced relaxations. Isolated bovine PCA were set up for measurement of isometric tension in organ baths containing oxygenated Krebs solution. The relaxant action of H2S donors was studied on phenylephrine-induced tone in the absence or presence of enzyme inhibitors for the following pathways: cyclooxygenase (COX); H2S; nitric oxide and the ATP-sensitive K(+) (KATP) channel. The H2S donors, NaSH (1 nM - 10 µM), AP67 (1 nM - 10 µM) and AP72 (10 nM - 1 µM) elicited a concentration-dependent relaxation of phenylephrine-induced tone in isolated bovine PCA. While the COX inhibitor, flurbiprofen (3 µM) blocked significantly (p < 0.05) the inhibitory response elicited by AP67, it had no effect on relaxations induced by NaSH and AP72. Both aminooxyacetic acid (30 µM) and propargylglycine (1 mM), enzyme inhibitors of H2S biosynthesis caused significant (p < 0.05) rightward shifts in the concentration-response curve to AP67 and AP72. Furthermore, the KATP channel antagonist, glibenclamide (300 µM) and the NO synthase inhibitor, l-NAME (100 µM) significantly attenuated (p < 0.05) the relaxation effect induced by AP67 and AP72 on PCA. We conclude that H2S donors can relax pre-contracted isolated bovine PCA, an effect dependent on endogenous production of H2S. The inhibitory action of only AP67 on pre-contracted PCA may involve the production of inhibitory endogenous prostanoids. Furthermore, the observed inhibitory action of H2S donors on PCA may depend on the endogenous biosynthesis of NO and by an action of KATP channels.

Role of the non-enzymatic metabolite of eicosapentaenoic acid, 5-epi-5-F3t-isoprostane in the regulation of [ (3)H]D-aspartate release in isolated bovine retina.

We have evidence that F2-isoprostanes (F2-IsoPs) regulate the release of excitatory neurotransmitters in isolated bovine retina. Although 5-F3-IsoPs are generated in mammals, in vivo, their pharmacological actions on neurotransmitter release remain unknown. In this study, we investigated the effect of 5-epi-5-F3t-IsoP on K(+)-evoked [(3)H]D-aspartate release in isolated bovine retina using the superfusion method. Furthermore, we examined the role of arachidonic acid metabolites in the regulation of the neurotransmitter release by this novel IsoP. In the concentration range, 0.01 nM-0.1 µM, 5-epi-5-F3t-IsoP inhibited K(+)-evoked [(3)H]D-aspartate release in a concentration-dependent manner, achieving a maximum inhibition of 46.9 % at 0.1 µM (IC30 = 1 nM). The prostanoid receptor antagonists, AH 6809 (EP1-3/DP; 10 µM), SC 51322 (EP1; 10 µM) and SC 19220 (EP1; 1 µM) partially reversed 5-epi-5-F3t-IsoP-mediated inhibition of K(+)-induced [(3)H]D-aspartate release. Pretreatment of retinal tissues with the cyclooxygenase (COX) inhibitor, flurbiprofen (3 µM) unmasked a biphasic action of 5-epi-5-F3t-IsoP that was inhibitory at lower (0.1-10 pM) and stimulatory at higher concentrations (≥0.1 nM). The prostanoid pathway antagonists, BAY-u3405 (10 µM; TP/DP-receptors), SQ 29548 (10 µM; TP-receptor) and ozagrel (10 µM; Tx-synthase inhibitor) abolished the stimulatory action of the 5-epi-5-F3t-IsoP (0.1 µM) on neurotransmitter release. In conclusion, 5-epi-5-F3t-IsoP attenuates K(+)-induced [(3)H]D-aspartate release in a concentration-dependent manner by mechanisms that are partially dependent on activation of pre-junctional prostanoid EP1-receptors. Moreover, blockade of the COX-pathway unmasks a biphasic action for 5-epi-5-F3t-IsoP that is inhibitory at low concentrations and stimulatory at higher concentrations. Products of the thromboxane synthase pathway may partially account for the stimulatory action of this F3-IsoP on isolated bovine retina.

Pharmacological actions of the slow release hydrogen sulfide donor GYY4137 on phenylephrine-induced tone in isolated bovine ciliary artery.

Hydrogen sulfide (H2S), a colorless gas characterized by its pungent odor of rotten eggs has been reported to elicit relaxation effects on basal and pre-contracted non-ocular smooth muscles of several mammalian species. In the present study, we investigated the pharmacological actions of a H2S donor, GYY4137 on isolated bovine posterior ciliary artery after contraction with the adrenergic receptor agonist, phenylephrine. Furthermore, we studied the underlying mechanism of inhibitory action of GYY4137 on the posterior ciliary arteries. Isolated bovine posterior ciliary arteries were mounted in oxygenated organ baths and changes in isometric tension were measured with a Grass FT03 transducer connected to a recorder using a Grass Polyview Software. The relaxant actions of GYY4137 on phenylephrine pre-contracted arteries were observed in the absence and presence of an inhibitor of cyclo-oxygenase, flurbiprofen. Furthermore, the inhibitory effects of GYY4137 were studied in the absence or presence of inhibitors/activators of biosynthetic enzymes for H2S and nitric oxide production, as well as specific ion channel blockers. In the concentration range, 100 nM to 100 µM, GYY4137 elicited a concentration-dependant relaxation of phenylephrine-induced tone in isolated posterior ciliary arteries, with IC50 value of 13.4 ± 1.9 µM (n = 6). The cyclo-oxygenase inhibitor, flurbiprofen, significantly (p < 0.01) enhanced the relaxation induced by GYY4137 yielding IC50 value of 0.13 ± 0.08 µM (n = 6). Both the inhibitors of cystathionine ß-synthase (aminooxyacetic acid, AOAA, 30 µM) and cystathionine γ-lyase (propargylglycine, PAG, 1 mM) caused significant (p < 0.05) rightward shifts in the concentration-response curve to GYY4137. Furthermore, the KATP channel antagonist, glibenclamide (100 µM) significantly (p < 0.01) attenuated the relaxant action induced by GYY4137 on bovine ciliary artery. Conversely, the activator of cystathionine ß-synthase, SAM (100 µM) and an inhibitor of nitric oxide synthase, L-NAME (100 µM) had no significant effect on relaxations induced by GYY4137. We conclude that the inhibitory action of GYY4137 on isolated bovine ciliary artery is dependent upon the endogenous production of both prostanoids and H2S. Furthermore, the observed vascular smooth muscle relaxation induced by GYY4137 is mediated, at least in part, by KATP channels.

Lipid peroxidation: pathophysiological and pharmacological implications in the eye.

Oxygen-derived free radicals such as hydroxyl and hydroperoxyl species have been shown to oxidize phospholipids and other membrane lipid components leading to lipid peroxidation. In the eye, lipid peroxidation has been reported to play an important role in degenerative ocular diseases (age-related macular degeneration, cataract, glaucoma, diabetic retinopathy). Indeed, ocular tissues are prone to damage from reactive oxygen species due to stress from constant exposure of the eye to sunlight, atmospheric oxygen and environmental chemicals. Furthermore, free radical catalyzed peroxidation of long chain polyunsaturated acids (LCPUFAs) such as arachidonic acid and docosahexaenoic acid leads to generation of LCPUFA metabolites including isoprostanes and neuroprostanes that may further exert pharmacological/toxicological actions in ocular tissues. Evidence from literature supports the presence of endogenous defense mechanisms against reactive oxygen species in the eye, thereby presenting new avenues for the prevention and treatment of ocular degeneration. Hydrogen peroxide (H2O2) and synthetic peroxides can exert pharmacological and toxicological effects on tissues of the anterior uvea of several mammalian species. There is evidence suggesting that the retina, especially retinal ganglion cells can exhibit unique characteristics of antioxidant defense mechanisms. In the posterior segment of the eye, H2O2 and synthetic peroxides produce an inhibitory action on glutamate release (using [(3)H]-D-aspartate as a marker), in vitro and on the endogenous glutamate and glycine concentrations in vivo. In addition to peroxides, isoprostanes can elicit both excitatory and inhibitory effects on norepinephrine (NE) release from sympathetic nerves in isolated mammalian iris ciliary bodies. Whereas isoprostanes attenuate dopamine release from mammalian neural retina, in vitro, these novel arachidonic acid metabolites exhibit a biphasic regulatory effect on glutamate release from retina and can regulate amino acid neurotransmitter metabolism without inducing cell death in the retina. Furthermore, there appears to be an inhibitory role for neuroprostanes in the release of excitatory amino acid neurotransmitters in mammalian retina. The ability of peroxides and metabolites of LCPUFA to alter the integrity of neurotransmitter pools provides new potential target sites and pathways for the treatment of degenerative ocular diseases.

Hydrogen sulfide: role in ion channel and transporter modulation in the eye.

Hydrogen sulfide (H(2)S), a colorless gas with a characteristic smell of rotten eggs, has been portrayed for decades as a toxic environmental pollutant. Since evidence of its basal production in mammalian tissues a decade ago, H(2)S has attracted substantial interest as a potential inorganic gaseous mediator with biological importance in cellular functions. Current research suggests that, next to its counterparts nitric oxide and carbon monoxide, H(2)S is an important multifunctional signaling molecule with pivotal regulatory roles in various physiological and pathophysiological processes as diverse as learning and memory, modulation of synaptic activities, cell survival, inflammation, and maintenance of vascular tone in the central nervous and cardiovascular systems. In contrast, there are few reports of a regulatory role of H(2)S in the eye. Accumulating reports on the pharmacological role of H(2)S in ocular tissues indicate the existence of a functional trans-sulfuration pathway and a potential physiological role for H(2)S as a gaseous neuromodulator in the eye. Thus, understanding the role of H(2)S in vision-related processes is imperative to our expanding knowledge of this molecule as a gaseous mediator in ocular tissues. This review aims to provide a comprehensive and current understanding of the potential role of H(2)S as a signaling molecule in the eye. This objective is achieved by discussing the involvement of H(2)S in the regulation of (1) ion channels such as calcium (L-type, T-type, and intracellular stores), potassium (K(ATP) and small conductance channels) and chloride channels, (2) glutamate transporters such as EAAT1/GLAST and the L-cystine/glutamate antiporter. The role of H(2)S as an important mediator in cellular functions and physiological processes that are triggered by its interaction with ion channels/transporters in the eye will also be discussed.