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We have evidence that F-isoprostanes (F-IsoPs) regulate the release of excitatory neurotransmitters in isolated bovine retina. Although 5-F-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-F-IsoP on K-evoked [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-F-IsoP inhibited K-evoked [H] d-aspartate release in a concentration-dependent manner, achieving a maximum inhibition of 46.9 % at 0.1 µM (IC = 1 nM). The prostanoid receptor antagonists, AH 6809 (EP/DP; 10 µM), SC 51322 (EP; 10 µM) and SC 19220 (EP; 1 µM) partially reversed 5- epi-5-F-IsoP-mediated inhibition of K-induced [H] d-aspartate release. Pretreatment of retinal tissues with the cyclooxygenase (COX) inhibitor, flurbiprofen (3 μM) unmasked a biphasic action of 5- epi-5-F-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-F-IsoP (0.1 μM) on neurotransmitter release. In conclusion, 5- epi-5-F-IsoP attenuates K-induced [H] d-aspartate release in a concentration-dependent manner by mechanisms that are partially dependent on activation of pre-junctional prostanoid EP-receptors. Moreover, blockade of the COX-pathway unmasks a biphasic action for 5- epi-5-F-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 F-IsoP on isolated bovine retina. [ABSTRACT FROM AUTHOR]

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 [3H]-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. [ABSTRACT FROM AUTHOR]

We have evidence that 15-F-isoprostanes (15-F-IsoPs) regulate excitatory neurotransmitter release in ocular tissues. Although 5-F-IsoPs are abundantly produced in mammals, their pharmacological actions on neurotransmitter release remain unknown. In the present study, we compared the effect of the 5-F-IsoP epimer pair, 5-F-IsoP (C5-OH in β-position) and 5- epi-5-F-IsoP (C5-OH in α-position), on K-evoked [H]D-aspartate release in isolated bovine retina. We further examined the role of prostanoid receptors on the inhibitory action of 5- epi-5-F-IsoP on [H]D-aspartate overflow. Isolated bovine retina were prepared for studies of K-evoked release of [H]D-aspartate using the superfusion method. 5- epi-5-F-IsoP (0.01 nM to 1 μM), attenuated K-evoked [H]D-aspartate release in a concentration-dependent manner, with the inhibitory effect of 26.9% ( P < 0.001; IC = 0.2 μM) being achieved at 1 μM concentration. Its 5-( S)-OH-epimer, 5-F-IsoP (0.1 nM-1 μM), exhibited an inhibitory biphasic action, yielding a maximal response of 35.7% ( P < 0.001) at 10 nM concentration of the drug (IC value of 3 nM). Although the prostanoid-receptor antagonists, AH 6809 (10 μM; EP/DP) and BAY-u3405 (10 μM; DP/Tx) exhibited no effect on 5- epi-5-F-IsoP (10 nM-1 μM)-mediated inhibition, SC-19220 (1 μM; EP) completely reversed 5- epi-5-F-IsoP (0.1 μM and 1 μM)-induced attenuation of K-evoked [H]D-aspartate release. Similarly, both SC-51322 (10 μM; EP) and AH 23848 (1 μM; EP) reversed the inhibitory action elicited by 5- epi-5-F-IsoP (0.1 μM) on the neurotransmitter release. We conclude that the 5-F-IsoP epimer pair, 5-F-IsoP and 5- epi-5-F-IsoP, attenuate K-induced [H]D-aspartate release in isolated bovine retina presumably via prostanoid receptor dependent mechanisms. The trans-orientation of the allylic hydroxyl group at position C5 accounts for the apparent biphasic response exhibited by 5-F-IsoP on excitatory neurotransmitter release. [ABSTRACT FROM AUTHOR]

The role of enzymes and receptors of the prostanoid pathway in the inhibitory effect of 8-isoprostaglandin E2 (8-isoPGE2) on endogenous amino acid neurotransmitter levels was examined, ex vivo. Freshly isolated bovine eyeballs were injected intravitreally with IsoPs, incubated in Krebs buffer for 30 min and retina prepared for HPLC-ECD detection of amino acids. 8-isoPGE2 attenuated retinal glutamate and its metabolite, glutamine and glycine in a concentration-dependent manner. The non-selective cyclooxygenase (COX)-inhibitor, flurbiprofen, COX-2 selective inhibitor, NS-398 and thromboxane (Tx) synthase inhibitor, furegrelate had no effect on both basal amino acid levels and the inhibitory effects of 8-isoPGE2 (1–100 μM) on the retinal amino acids. Whereas the TP-receptor antagonist SQ-29548(10 μM) exhibited no effect, SC-19220(EP1; 30 μM), AH-6809(EP1–3; 30 μM) and AH-23848(EP4; 30 μM) reversed the inhibitory effects of 8-isoPGE2 (0.01–100 μM) on glutamate, glutamine and glycine levels. We conclude that prostanoid EP-receptors regulate the inhibitory effect of 8-isoPGE2 on basal levels of endogenous amino acids in bovine retina, ex vivo. [ABSTRACT FROM AUTHOR]

Hydrogen sulfide (H2S), can produce pharmacological effects on neural and non-neural tissues from several mammalian species. The present study investigates the pharmacological action of H2S, (using sodium hydrosulfide, NaHS, and/or sodium sulfide, Na2S as donors) on amino acid neurotransmission (using [3H] d-aspartate as a marker for glutamate) from isolated, superfused bovine and porcine retinae. Isolated neural retinae were incubated in Krebs solution containing [3H] d-aspartate at 37°C. Release of [3H] d-aspartate was elicited by high potassium (K+ 50 mM) pulse. Both NaHS and Na2S donors caused an inhibition of K+-evoked [3H] d-aspartate release from isolated bovine retinae without affecting basal [3H] d-aspartate efflux yielding IC50 values of 0.006 and 6 μm, respectively. Furthermore, NaHS inhibited depolarization-evoked release of [3H] d-aspartate from isolated porcine retinae with an IC50 value of 8 μM. The inhibitory action of NaHS on [3H] d-aspartate release from porcine retinae was blocked by propargyglycine, a selective inhibitor of cystathionine γ-lyase (CSE). Our results indicate that H2S donors can inhibit amino acid neurotransmission from both isolated bovine and porcine retinae, an effect that is dependent, at least in part, on intramural biosynthesis of H2S. [ABSTRACT FROM AUTHOR]

We investigated the effect of cannabinoids on potassium chloride (K + )- and ischemia-induced [ 3 H]D-aspartate release from isolated bovine retinae. The superfusion method was employed for studies of [ 3 H]-neurotransmitter release. Cannabinoid receptor CB1 agonists, but not the CB2 agonist JWH 015, inhibited K + -induced [ 3 H]D-aspartate release from bovine retinae with the following rank order of activity: anandamide > ACEA > methanandamide > WIN 55,212-2. In the ischemic model, the rank order of activity was as follows: methanandamide > ACEA > WIN 55,212-2. The CB1 receptor antagonist AM 251 blocked inhibitory responses produced by cannabinoids in both experimental conditions. In conclusion, cannabinoids inhibit evoked [ 3 H]D-aspartate release from isolated bovine retinae via an effect on CB1 receptors. [ABSTRACT FROM AUTHOR]

We have previously shown that hydrogen peroxide (H2O2) can inhibit K -depolarization-evoked [²H ]D-aspartate release from bovine isolated retinae. In the present study, we investigated the role of arachidonic acid metabolites in the inhibitory response elicited by H2O2 in the bovine retinae. Furthermore, we examined the direct effect of H2O2 on the production of prostaglandins and isoprostanes in this tissue. Isolated bovine retinae were prepared for studies of [³H]D- aspartate release using tile Superfusion Method. Release of [&sup3]D-aspartate was elicited by Krebs solution containing an iso-osmotic concentration of KCl (50mM). A direct action of H2O2 on prostaglandin E2 (PGE2) and 8-isoprostane F2α (8-iso-PGF2α) was also measured by enzyme-linked immunosorbant assay (ELISA). The cyclooxygenase inhibitor, flurbiprofen (3μM), or the thromboxane-receptor antagonist. SQ 29548 (10μM) had no significant (p > 0.05) effect on K-evoked [²H]D-aspartate release. On the other hand, both flurbiprofen (3μM) and SQ 29548 (10μM) blocked the inhibition of K-evoked [&sup3H]D-aspartate induced by H2O2: (30μM). In concentrations up to 100μM. H2O2 caused an increase in PGE2 and 8-iso-PGF2α over basal levels. For instance. H2O2. (100μM) increased PGE2 and 8 iso-PGF2α over basal levels by 348 ± 41% and 185 ± 26 (n= 4), respectively. We conclude that the peroxide-mediated inhibition of [³H]D-aspartate may involve the production of prostaglandins and isoprostanes in the bovine isolated retinae. [ABSTRACT FROM AUTHOR]

The glucose deprivation-induced release of [[sup 3]H]D-aspartate was studied in bovine and human retinas in a superfusion apparatus. [[sup 3]H]D-aspartate release was significantly increased upon omitting glucose in the superfusion buffer. This effect was dependent on external Ca[sup 2+] because L- and N-type Ca[sup 2+]-channel blockers, such as diltiazem (1 μM), nitrendipine (1 μM), and ω-conotoxin (100 nM), significantly reduced the effect of glucose-deprivation induced release of [[sup 3]H]D-aspartate. Furthermore, while glutamate receptor agonists (L-glutamate, N-methyl-D-aspartate, but not kainate) potentiated the effects of glucose deprivation, antagonists (MK-801, MCPG, ifenprodil, and L-AP3) at these receptors blocked the glucose deprivation-induced release process. Taken together, these studies have demonstrated that under conditions of glucose deprivation, as may happen during ischemic events in vivo, the retinal glutamatergic nerve endings and/or glial cells promote the efflux of [[sup 3]H]D-aspartate into the extracellular environment. This process appears to be receptor-mediated and dependent on extracellular Ca[sup 2+] and is similar to previous reports pertaining to brain tissues. [ABSTRACT FROM AUTHOR]

Purpose. The aim of the present study was two-fold: (a) to examine the effect of hypoxia on [³H]D-aspartate release from isolated bovine and human retinae, and (b) to investigate the regulation of hypoxia-induced neurotransmitter release by glutamate receptor agonists and antagonists. Methods. Isolated neural retinae were incubated in oxygenated Krebs buffer solution containing [³H]D-aspartate and then prepared for studies of neurotransmitter release using the superfusion method. Release of [³H]D-aspartate was evoked by K[sup +] (50mM) applied at 90 minutes (S[sub 1]) and hypoxia (induced by exposure of tissues to solutions pregassed with 95% N[sub 2]: 5% CO[sub 2] for 60 minutes) at 108 minutes (S[sub 2]) after onset of superfusion. Results. Under hypoxic conditions, pO[sub 2] in normal Krebs buffer solution was reduced from 14.53 ± 0.26 ppm (n = 6) to 0.54 ± 0.04 ppm (n = 9) after one hour of gassing with 95% N[sub 2]: 5% CO[sub 2]. Exposure to hypoxia elicited an overflow of [³H]D-aspartate yielding S[sub 2]/S[sub 1] ratios of 0.62 ± 0.06 (n = 12) and 0.54 ± 0.03 (n = 8) in bovine and human tissues respectively. In isolated bovine retinae, L- and N-calciumchannel antagonists diltiazem, nitrendipine, verapamil and ω-conotoxin significantly (p < 0.01 or higher) attenuated hypoxia-induced [³H]D-aspartate release. L-glutamate (30 µM) significantly (p < 0.001) potentiated hypoxiainduced [³H]D-aspartate release whereas kainate (30 µM) inhibited this response. NMDA (in concentrations up to 1 mM) had no effect on hypoxia-induced [³H]D-aspartate release. Antagonists of glutamate receptors and the polyamine site on the NMDA receptor inhibited hypoxia-induced release of [³H]D-aspartate in bovine retina with the following rank order of activity: ifenprodil ≅ MCPG > L-AP3 > MK-801. At an equimolar concentration (10 µM), L-AP3 but not ifenprodil, MCPG, MK 801 or arcaine,... [ABSTRACT FROM AUTHOR]

Peroxides can enhance field-stimulated [3H]norepinephrine ([3H]NE) release in isolated irides from several mammalian species. In the present study, we investigated the role of prejunctional α2-adrenoceptors in peroxide-induced potentiation of sympathetic neurotransmission in bovine isolated irides. Isolated hemi-irides were incubated in a Krebs buffered-solution containing [3H]NE and prepared for studies of neurotransmitter release using the superfusion method. α2-Adrenoceptor agonists, oxymetazoline, UK-14304 and clonidine inhibited field-stimulated [3H]NE overflow without affecting basal tritium efflux. Pretreatment of tissues with H2O2 (300 μM) had no effect on inhibition of evoked [3H]NE release caused by the α2-adrenergic agonists. However, H2O2 (300 μM) caused significant (P < 0.01) leftward shifts of excitatory concentration-response curves to yohimbine (10 nM–1 μM). In contrast, yohimbine (1 μM) did not prevent the enhancement of evoked [3H]NE overflow induced by H2O2 (300 μM). In conclusion, excitatory effects of peroxides on sympathetic neurotransmission in bovine irides are not mediated by prejunctional α2-adrenoceptors. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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-β-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. [ABSTRACT FROM AUTHOR]

Background: Hydrogen sulfide (H2S)-releasing compounds can reduce intraocular pressure in normotensive rabbits by increasing aqueous humor (AH) outflow through the trabecular meshwork. In the present study, we investigated the contribution of endogenous H2S and the role of intramurally generated prostaglandins in the observed increase in AH outflow facility in an ex vivo porcine ocular anterior segment model. Material and Methods: Porcine ocular anterior segment explants were perfused with Dulbecco's Modified Eagle's Medium maintained at 37 °C and gassed with 5% CO2 and 95% air under an elevated pressure of 15 mmHg for four hours. Perfusates from the anterior segment explants were collected and immediately assayed for their H2S and prostaglandin E2 content. Results: Elevating perfusion pressure from 7.35 to 15 mm Hg significantly (p < 0.001) increased H2S concentration in the perfusate from 0.4 ± 0.1 to 67.6 ± 3.6 nM/µg protein. In the presence of an inhibitor of cystathionine ß-synthase/cystathionine γ-lyase, aminooxyacetic acid (AOAA, 30 µM), or an inhibitor of 3-mercaptopyruvate sulfurtransferase, α-ketobutyric acid (KBA, 1 mM), the effects of elevated pressure on H2S levels in the perfusate was significant (p < 0.001). Furthermore, flurbiprofen (30 µM) and indomethacin (10 µM) attenuated the elevated pressure-induced increase in H2S levels in the perfusate. Interestingly, elevating perfusion pressure had no significant effect on PGE2 concentrations in the perfusate. While the inhibition of H2S biosynthesis by AOAA or KBA did not affect PGE2 levels in perfusate, flurbiprofen (30 µM) caused a significant (p < 0.05) decrease in the concentration of PGE2 under conditions of elevated perfusion pressure. Conclusions: We conclude that the elevated perfusion pressure-induced increase in H2S concentrations depends upon the endogenous biosynthesis of H2S and intramurally produced prostaglandins in the porcine anterior segment explants. While the concentration of PGE2 in the perfusate under elevated perfusion pressure was unaffected by pretreatment with inhibitors of H2S biosynthesis, it was reduced in the presence of an inhibitor of cyclooxygenase. [ABSTRACT FROM AUTHOR]

Cataract, one of the leading causes of blindness worldwide, is a condition in which complete or partial opacity develops in the lens of the eyes, thereby impairing vision. This study aimed to examine the potential therapeutic and protective effects of poorly soluble polyphenols like curcumin, resveratrol, and dibenzoylmethane, known to possess significant antioxidant activity. The polyphenols were loaded into novel lipid-cyclodextrin-based nanoparticles and characterized by particle size, polydispersity index, differential scanning calorimetry, thermogravimetric analysis, X-ray diffraction, scanning electron microscopy (SEM), entrapment efficiency, and release studies. Ferric-reducing ability of plasma and 2,2-diphenyl-1-picrylhydrazyl chemical assays were used to evaluate their antioxidant properties based on their free radical quenching ability. Biochemical in vitro assays were used to examine these polyphenols on hydrogen peroxide-induced formation of cataracts in bovine lenses by estimating total glutathione content and superoxide dismutase activity. Nanoparticles were thermostable and amorphous. Particle size of curcumin, resveratrol, and dibenzoylmethane nanoparticles were 331.0 ± 17.9 nm, 329.9 ± 1.9 nm, and 163.8 ± 3.2 nm, respectively. SEM confirmed porous morphology and XRD confirmed physical stability. Entrapment efficiency for curcumin-, resveratrol-, and dibenzoylmethane-loaded nanoparticles was calculated to be 84.4 ± 2.4%, 72.2 ± 1.5%, and 86.4 ± 0.6%, respectively. In vitro release studies showed an initial burst release followed by a continuous release of polyphenols from nanoparticles. Chemical assays confirmed the polyphenols' antioxidant activity. Superoxide dismutase and glutathione levels were found to be significantly increased (p < 0.05) after treatment with polyphenol-loaded nanoparticles than pure polyphenols; thus, an improved antioxidant activity translational into potential anticataract activity of the polyphenols when loaded into nanoparticles was observed as compared to pure polyphenols. [ABSTRACT FROM AUTHOR]

Abstract: 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 β-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 β-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. [Copyright &y& Elsevier]

Abstract: Hydrogen sulfide (H2S), a colorless gas with the pungent odor of rotten eggs has been reported to produce pharmacological actions in ocular and non-ocular tissues. We have evidence that H2S, using sodium hydrosulfide (NaHS) and sodium sulfide (Na2S) as donors can increase cyclic AMP (cAMP) production in neural retina. In the present study, we investigated the mechanism of action of H2S on cyclic nucleotide production in rat retinal pigment epithelial cells (RPE-J). Cultured RPE-J cells were incubated for 30min in culture medium containing the cyclic nucleotide phosphodiesterase (PDE) inhibitor, IBMX (2mM). Cells were exposed to varying concentrations of NaHS, the H2S substrate (l-cysteine), cyclooxygenase (COX) inhibitors or the diterpene activator of adenylate cyclase, forskolin in the presence or absence of H2S biosynthetic enzymes or the ATP-sensitive potassium (KATP) channel antagonist, glibenclamide. Following drug-treatment at different time intervals, cell homogenates were prepared for cAMP assay using a well established methodology. In RPE-J cells, NaHS (10nM–1μM) produced a time-dependent increase in cAMP concentrations over basal levels which reached a maximum at 20min. At this time point, both NaHS (1nM–100μM) and l-cysteine (1nM–10μM) produced a concentration-dependent significant (p <0.05) increase in cAMP concentrations over basal level. The effects of NaHS on cAMP levels in RPE-J cells was enhanced significantly (p <0.01) in the presence of the COX inhibitors, indomethacin and flurbiprofen. In RPE-J cells, the effects caused by forskolin (10μM) on cAMP production were potentiated by addition of low concentrations of NaHS. Both the inhibitor of cystathionine β-synthase (CBS), aminooxyacetic acid (AOA, 1mM) and the inhibitor of cystathionine γ-lyase (CSE), proparglyglycine (PAG, 1mM) significantly attenuated the increased effect of l-cysteine on cAMP production. The KATP channel antagonist, glibenclamide (100μM) caused inhibition of NaHS induced-increase of cAMP formation in RPE-J cells. We conclude that, H2S (using H2S donor and substrate) can increase cAMP production in RPE-J cells, and removal of the apparent inhibitory effect of prostaglandins unmasks an excitatory activity of H2S on cAMP. Effects elicited by the H2S substrate on cAMP formation are dependent on biosynthesis of H2S catalyzed by the biosynthetic enzymes, CBS and CSE. In addition to the adenylyl cylcase pathway, KATP channels are involved in mediating the observed effects of the H2S on cAMP production. [Copyright &y& Elsevier]

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

In the present study, we investigate the inhibitory effect of novel H 2 S 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 H 2 S donors was studied on phenylephrine-induced tone in the absence or presence of enzyme inhibitors for the following pathways: cyclooxygenase (COX); H 2 S; nitric oxide and the ATP-sensitive K + (K ATP ) channel. The H 2 S 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 H 2 S biosynthesis caused significant ( p < 0.05) rightward shifts in the concentration–response curve to AP67 and AP72. Furthermore, the K ATP 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 H 2 S donors can relax pre-contracted isolated bovine PCA, an effect dependent on endogenous production of H 2 S. The inhibitory action of only AP67 on pre-contracted PCA may involve the production of inhibitory endogenous prostanoids. Furthermore, the observed inhibitory action of H 2 S donors on PCA may depend on the endogenous biosynthesis of NO and by an action of K ATP channels. [ABSTRACT FROM AUTHOR]

Highlights: [•] Total synthesis report of isoprostanes (IsoPs) and neuroprostanes (NeuroPs). [•] IsoPs and NeuroPs derived from a non-enzymatic peroxidation of PUFAs bound to phospholipids. [•] IsoPs are the most reliable biomarkers of lipid peroxidation in humans and animal models. [•] Several IsoPs have also been shown to be biologically active. [ABSTRACT FROM AUTHOR]

Hydrogen sulfide (HS) is a novel gasotransmitter with physiological and pathological functions in vascular homeostasis, cardiovascular system and central nervous system. In the present study, we determined the endogenous levels of HS in various tissues of the bovine eye. We also examined the basal levels of HS in response to donors (sodium hydrosulfide, NaHS and sodium sulfide, NaS), substrate ( l-cysteine), inhibitors (propargylglycine, PAG and aminooxyacetic acid, AOA) and activator (S-adenosyl- l-methionine, SAM) of this gas in the bovine retina. HS was measured using a well established spectrophotometric method. The highest concentration of endogenous HS was detected in cornea (19 ± 2.85 nmoles/mg protein, n = 6) and retina (17 ± 2.1 nmoles/mg protein, n = 6). Interestingly, HS was not present in vitreous humor. The inhibitors of CSE and CBS; PAG (1 mM) and AOA (1 mM), significantly attenuated the production of HS in the bovine retina by 56.8 and 42%, respectively. On the other hand the activator of CBS; SAM (100 μM), HS donors; NaHS (1 μM) and NaS (100 μM), significantly increased endogenous levels of HS in bovine retina. l-cysteine (10-300 μM) produced a significant ( P < 0.05) concentration-dependent increase in HS levels reaching a maximal at 300 μM. We conclude that HS is endogenously produced in various tissues of the isolated bovine eye. Moreover, endogenous levels of HS are enhanced in the presence of substrate ( l-cysteine), an activator of CBS (SAM) and HS donors but are blocked by inhibitors of enzymes that synthesize this gas in neural retina. [ABSTRACT FROM AUTHOR]