Regulation of [3H] D-aspartate release from mammalian isolated retinae by hydrogen sulfide.

Hydrogen sulfide (H(2)S), can produce pharmacological effects on neural and non-neural tissues from several mammalian species. The present study investigates the pharmacological action of H(2)S, (using sodium hydrosulfide, NaHS, and/or sodium sulfide, Na(2)S as donors) on amino acid neurotransmission (using [(3)H] D: -aspartate as a marker for glutamate) from isolated, superfused bovine and porcine retinae. Isolated neural retinae were incubated in Krebs solution containing [(3)H] D: -aspartate at 37 degrees C. Release of [(3)H] D: -aspartate was elicited by high potassium (K(+) 50 mM) pulse. Both NaHS and Na(2)S donors caused an inhibition of K(+)-evoked [(3)H] D: -aspartate release from isolated bovine retinae without affecting basal [(3)H] D: -aspartate efflux yielding IC(50) values of 0.006 and 6 microm, respectively. Furthermore, NaHS inhibited depolarization-evoked release of [(3)H] D: -aspartate from isolated porcine retinae with an IC(50) value of 8 microM. The inhibitory action of NaHS on [(3)H] D: -aspartate release from porcine retinae was blocked by propargyglycine, a selective inhibitor of cystathionine gamma-lyase (CSE). Our results indicate that H(2)S 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 H(2)S.

Effect of hydrogen sulfide on sympathetic neurotransmission and catecholamine levels in isolated porcine iris-ciliary body.

In the present study, we investigated the pharmacological action of hydrogen sulfide (H2S, using sodium hydrosulfide, NaHS, and/or sodium sulfide, Na2S as donors) on sympathetic neurotransmission from isolated, superfused porcine iris-ciliary bodies. We also examined the effect of H2S on norepinephrine (NE), dopamine and epinephrine concentrations in isolated porcine anterior uvea. Release of [3H]NE was triggered by electrical field stimulation and basal catecholamine concentrations was measured by high performance liquid chromatography (HPLC). Both NaHS and Na2S caused a concentration-dependent inhibition of electrically evoked [3H]NE release from porcine iris-ciliary body without affecting basal [3H]NE efflux. The inhibitory action of H2S donors on NE release was attenuated by aminooxyacetic acid (AOA) and propargyglycine (PAG), inhibitors of cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE), respectively. With the exception of dopamine, NaHS caused a concentration-dependent reduction in endogenous NE and epinephrine concentrations in isolated iris-ciliary bodies. We conclude that H2S can inhibit sympathetic neurotransmission from isolated porcine anterior uvea, an effect that is dependent, at least in part, on intramural biosynthesis of this gas. Furthermore, the observed action of H2S donors on sympathetic transmission may be due to a direct action of this gas on neurotransmitter pools.

Inhibitory action of hydrogen sulfide on muscarinic receptor-induced contraction of isolated porcine irides.

We investigated the pharmacological actions of hydrogen sulfide (H(2)S) using sodium hydrosulfide (NaHS) and sodium sulfide (Na(2)S) as donors on isolated porcine irides in the presence of tone induced by muscarinic receptor stimulation. Furthermore, we also investigated the mechanism of action of H(2)S in this smooth muscle. Isolated porcine iris muscle strips were set up in organ baths and prepared for measurement of longitudinal isometric tension. The relaxant action of NaHS or Na(2)S on carbachol-induced tone was studied in the absence and presence of a K(+)-channel inhibitor and inhibitors/activators of enzymes of the biosynthetic pathways for H(2)S, prostanoid and nitric oxide production. In the concentration range, 10 nM to 100 microM, NaHS produced a concentration-dependent relaxation of carbachol-induced tone reaching a maximum of inhibition of 28% at 30 microM. The cyclooxygenase inhibitor, flurbiprofen (1 microM), enhanced relaxations induced by both NaHS and Na(2)S yielding IC(50) values of 7 microM and 70 microM, respectively. With exception of l-NAME (300 muM) inhibitors of cystathionine gamma-lyase, propargylglycine, (PAG) (1 mM) and beta-cyanoalanine, (BCA) (1 mM) and inhibitors of cystathionine beta-synthase, aminooxyacetic acid (AOA) (30 microM) and hydroxylamine (HOA) (30 microM) caused significant (P < 0.001) rightward shifts in the concentration-response curves to NaHS. An activator of cystathionine beta-synthase, SAM (100 microM), enhanced relaxations elicited by low concentrations of NaHS but attenuated responses caused by the higher concentrations of this H(2)S donor. The inhibitor of K(ATP) channel, glibenclamide (100 and 300 microM), blocked relaxations induced by NaHS. We conclude that the observed inhibitory action of NaHS and Na(2)S in isolated porcine irides is dependent on endogenous production of prostanoids and the biosynthesis of H(2)S by cystathionine gamma-lyase and cystathionine beta-synthase. Furthermore, relaxation induced by H(2)S is mediated, at least in part, by K(ATP) channels. Nitric oxide is not involved in the relaxation induced by this gas in the isolated porcine irides.

Aqueous humor outflow effects of 2-arachidonylglycerol.

This study was conducted to test the effects of 2-arachidonylglycerol (2-AG), an endocannabinoid, on aqueous humor outflow facility, to study the cellular mechanisms of 2-AG, and to investigate the possible existence and activity of monoacylgylcerol lipase (MGL), a 2-AG metabolic enzyme, in the trabecular meshwork (TM). The effects of 2-AG on aqueous humor outflow facility were measured using an anterior segment perfused organ culture model. The expression and activity of MGL in TM tissues were assessed using Western blot analysis and an enzyme activity assay respectively. 2-AG induced activation of p42/44 mitogen-activated protein (MAP) kinase was determined by Western blot analysis using an anti-phospho p42/44 MAP kinase antibody. AlexaFluor 488-labeled phalloidin staining was used to examine actin filament in cultured TM cells. Administration of 10nM of 2-AG caused a transient enhancement of aqueous humor outflow. In the presence of 100nM of LY2183240, an inhibitor of MGL, the effect of 10nM of 2-AG on outflow was prolonged by at least 4h. The 2-AG-induced enhancement of outflow was blocked by SR141716A, a CB1 antagonist, and SR144528, a CB2 antagonist. In Western blot studies, a 35kDa band representing MGL was detected on TM tissues with an anti-MGL antibody. The 2-AG enzymatic hydrolysis activity was detected in TM tissues and this activity was reduced by 70.1+/-5.3% with the addition of 100 nM of LY2183240. Treatment of trabecular meshwork cells with 10nM of 2-AG plus 100 nM LY2183240 for 5h evoked phosphorylation of p42/44 MAP kinase. The 2-AG-induced enhancement of p42/44 MAP kinase phosphorylation was blocked by pretreatment with SR141716A, SR144528, as well as PD98059, an inhibitor of the p42/44 MAP kinase pathway. In addition, the outflow-enhancing effect of 2-AG was blocked by pretreatment with PD98059. Furthermore, treatment with 2-AG plus LY2183240 caused rounding of TM cells and a reduction of actin stress fibers in TM cells. Pretreatment with SR141716A, SR144528, and PD98059 blocked these 2-AG-induced morphology and cytoskeleton changes in TM cells. In conclusion, the results from this study demonstrate that administration of 2-AG increases aqueous humor outflow facility and this effect of 2-AG is mediated through both the CB1 and CB2 cannabinoid receptors. In addition, this study reveals the existence and the activity of MGL, a 2-AG metabolizing enzyme, in the TM tissues. Furthermore, this study suggests that 2-AG-induced enhancement of outflow facility involves the p42/44 MAP kinase signaling pathway and changes in actin cytoskeletons in TM cells.

N-arachidonylethanolamide-induced increase in aqueous humor outflow facility.

PURPOSE: To study the effects of N-arachidonylethanolamide (anandamide [AEA]) on aqueous humor outflow and to investigate the existence and activity of fatty acid amide hydrolase (FAAH), an AEA metabolic enzyme in trabecular meshwork (TM) tissue. METHODS: The effects of AEA on aqueous humor outflow were measured using a porcine anterior segment-perfused organ culture model. Western blot analysis was used to study the expression of FAAH, and a thin-layer chromatography-based approach was used to measure the enzymatic activity of FAAH in TM tissue. RESULTS: Administration of AEA caused a transient enhancement of aqueous humor outflow facility. In the presence of 100 nM URB597, an FAAH inhibitor, the effect of 10 nM AEA on outflow facility was prolonged by at least 4 hours. The AEA-induced enhancement of outflow facility was blocked by SR141716A, a CB1 antagonist, and was partially blocked by SR144528, a CB2 antagonist. In Western blot studies, positive signals were detected on TM tissues with an anti-FAAH antibody. In the enzyme activity studies, the enzymatic activity of AEA hydrolysis was detected in TM tissues, and this activity was reduced with the addition of 100 nM URB597. CONCLUSIONS: Results from this study demonstrate that the administration of AEA increases aqueous humor outflow facility and that this effect of AEA involves CB1 and CB2 cannabinoid receptors. In addition, this study reveals the existence and the activity of FAAH, an AEA-metabolizing enzyme, in the TM tissues.

Noladin ether acts on trabecular meshwork cannabinoid (CB1) receptors to enhance aqueous humor outflow facility.

PURPOSE: To study the effects of 2-arachidonyl glyceryl ether (noladin ether), an endocannabinoid ligand selective for cannabinoid (CB)1 receptor, on aqueous humor outflow facility, to investigate the involvement of trabecular meshwork CB1 receptors and the p42/44 MAP kinase signaling pathway and to explore the cellular mechanisms of noladin ether-induced changes of outflow facility. METHODS: The effects of noladin ether on aqueous humor outflow facility were measured in a porcine anterior-segment-perfused organ culture model. The expression of CB1 receptors on cultured porcine trabecular meshwork cells and the coupling of these receptors to p42/44 MAP kinase was determined by immunofluorescence microscopy and Western blot analysis. Both Western blot and zymography were used to monitor the effects of noladin ether on matrix metalloproteinase (MMP)-2. In morphologic studies, AlexaFluor 488-labeled phalloidin staining was used to examine actin filament, and immunohistochemistry with anti-paxillin antibodies was used to detect focal adhesions. RESULTS: Within 1 hour after adding 3, 30, or 300 nM of noladin ether, the aqueous humor outflow facility increased concentration dependently. The effect of 30 nM of noladin ether was completely blocked by SR141716A, a selective CB1 antagonist. Positive signals were detected on cultured porcine trabecular meshwork cells with an anti-CB1 antibody in immunofluorescence microscopy and Western blot studies. Treatment of trabecular meshwork cells with 30 nM of noladin ether activated p42/44 MAP kinase, whereas pretreatment with SR141716A blocked the p42/44 MAP kinase-activating effects of noladin ether. In addition, the enhancement of outflow facility induced by noladin ether was blocked by pretreatment of porcine anterior segments with PD98059, an inhibitor of p42/44 MAP kinase pathway. Furthermore, noladin ether treatment caused rounding of trabecular meshwork cells, and there was a decrease of actin stress fibers, as well as a decrease in focal adhesions. These noladin ether-induced morphologic changes were also blocked by SR141716A and PD98059. CONCLUSIONS: The results demonstrate for the first time that administration of noladin ether, an endocannabinoid agonist selective for the CB1 receptor, increases aqueous humor outflow facility. The data also show that noladin ether-induced enhancement of outflow facility is mediated through the trabecular meshwork CB1 receptor, with an involvement of p42/44 MAP kinase signaling pathway and changes in actin cytoskeletons.