cAMP-responsive element binding protein mediates a cGMP/protein kinase G-dependent anti-apoptotic signal induced by nitric oxide in retinal neuro-glial progenitor cells

Nitric oxide (NO) is cytoprotective to certain types of neuronal cells. The neuroprotective ability of NO in the retina was reportedly mediated by the cyclic GMP (cGMP) to protein kinase G (PKG) pathway. Cyclic AMP-responsive element binding protein (CREB) plays an essential role in the NO/cGMP/PKG-mediated survival of rat cerebellar granule cells. We tested whether CREB transduces the NO/cGMP/PKG anti-apoptotic cascade in R28 neuro-glial progenitor cells. Apoptosis was induced in R28 cells by serum deprivation for 24 h. Varying concentrations of two NO donors, sodium nitroprusside (SNP) and nipradilol, were added to medium with or without an NO scavenger, a soluble guanylyl cyclase inhibitor, or a PKG inhibitor. The cells were immunostained against activated caspase-3 and counterstained with Hoechst 33258. Apoptosis was quantified by counting activated caspase-3 positive or pyknotic cells. SNP and nipradilol rescued R28 cells from apoptosis in a dose-dependent manner, at an optimal concentration of 1.0 microM and 10 microM, respectively. Higher concentrations were cytotoxic. The NO scavenger and the inhibitors decreased the anti-apoptotic effect of the NO donors. Intracellular cGMP levels were increased after exposure to SNP and nipradilol. Western blotting showed that both NO donors increased CREB phosphorylation, which was blocked when pre-exposed to the inhibitors. Transfection with a dominant negative CREB construct defective of phosphorylation at Ser-133 interfered with the anti-apoptotic activity of SNP. These results indicate that CREB at least in part mediates the cGMP/PKG-dependent anti-apoptotic signal induced by NO in R28 cells.     

Localization of heme oxygenase-2 and modulation of cGMP levels by carbon monoxide and/or nitric oxide in the retina

Heme oxygenase-2 (HO-2) synthesizes carbon monoxide (CO), a modulator of soluble guanylate cyclase (sGC). To examine this signal transduction pathway in the retina, we immunocytochemically localized HO-2, and investigated the effects of CO on cGMP levels. In turtle, HO-2-like immunoreactivity (-LI) was in all photoreceptors, some amacrine cells, and in numerous bipolar and ganglion cells. HO-2-LI colocalized with sGC activity in many cells. In rat, HO-LI was found only in the inner retina, in ganglion and amacrine cells. In turtle, stimulation with CO alone primarily increased cGMP-LI in bipolar cells in the visual streak. Stimulation with a combination of CO and nitric oxide (NO) dramatically increased cGMP-LI throughout the retina, in comparison to the smaller increases seen with NO or CO alone. These data suggest that CO is an endogenous modulator of the sGC/cGMP signaling pathway in many retinal neurons, and can dramatically amplify NO-stimulated increases in cGMP.     

Effect of shear stress on the hydraulic conductivity of cultured bovine retinal microvascular endothelial cell monolayers

The shear stress of flowing blood on endothelial cells increases water transport (hydraulic conductivity, Lp) in several vascular beds in vivo and has been hypothesized to play a role in elevating vascular transport in ocular diseases such as diabetic retinopathy. The purpose of this study is to determine the response of Lp to varying levels of shear stress using an in vitro model of the blood-retinal barrier: bovine retinal endothelial cells (BRECs) grown on polycarbonate filters. The study also addresses the role of nitric oxide (NO) and other downstream effectors in mediating shear-induced changes in water transport. A step change in shear stress of 10 dyn/cm(2) did not produce a significant change in Lp over 3 hours, whereas a 20 dyn/cm(2) step change elevated Lp by 14.6-fold relative to stationary controls at the end of 3h of shear exposure. 20 dyn/cm( 2) of shear stress stimulated the endothelial monolayers to release nitric oxide in a biphasic manner and incubation of the BRECs with a nitric oxide synthase (NOS) inhibitor, L-NMMA, significantly attenuated the shear-induced Lp response. These experiments demonstrate that NO is a key signaling molecule in the pathway linking shear stress and Lp in BRECs. A widely studied pathway downstream of NO involves the activation of guanylate cyclase (GC), guanosine 3', 5' -- cyclic monophosphate (cGMP) and protein kinase G (PKG). It was observed that incubation of BRECs with the GC inhibitor, LY83583 (10 microM) or the PKG inhibitor, KT5823 (1 microM) did not significantly alter the shear-induced Lp response. Also the cGMP analogue, 8-br-cGMP (1mM), did not affect the baseline Lp over 4h. These results demonstrate that shear stress elevates hydraulic conductivity in BRECs through a signaling mechanism that involves NO but not the GC/cGMP/PKG pathway.     

Nitric oxide donor stimulated increase of cyclic GMP in the goldfish retina

Nitric oxide (NO) activates soluble guanylyl cyclase (sGC) and the resulting increase in cyclic guanosine monophosphate (cGMP) is an important intracellular signalling pathway in the vertebrate retina. Immunocytochemical detection of cGMP following exposure to NO donors has proven an effective method of identifying cells that express sGC. While such an approach has proven useful for the study of several vertebrate retinas, it has not been applied to the well-characterized teleost retina. Therefore, in the present study, we have applied this approach to the retina of the goldfish (Carassius auratus). In the presence of the phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX), incubation of goldfish eyecups in Ringer's solution containing (+/-)-S-nitroso-N-acetylpenicillamine (SNAP) increased cGMP-like immunoreactivity (cG-ir) in bipolar, horizontal, amacrine, and ganglion cells and in ganglion cell axons and optic nerve. Weak labeling was observed in horizontal cells but no change in cG-ir was noted within photoreceptors. The NO donor-stimulated increases of cG-ir in horizontal, bipolar, amacrine, and ganglion cells are consistent with known physiological effects of NO on these neurons. The physiological significance of NO action at the level of optic nerve is not known. The lack of an effect of SNAP on cG-ir in photoreceptors was unexpected, as there are known physiological actions of NO, mediated by cGMP, on these neurons. Although this may be due to insufficient sensitivity of immunolabeling, this result may indicate a difference between isoforms of sGC or cGMP PDE in these neurons, compared to neurons where exogenous NO increased cG-ir.     

Gycine and GABA interact to regulate the nitric oxide/cGMP signaling pathway in the turtle retina

Nitric oxide (NO) is a free radical that is important in retinal signal transduction and cyclic guanosine monophosphate (cGMP) is a critical downstream messenger of NO. The NO/cGMP signaling pathway has been shown to modulate neurotransmitter release and gap junction coupling in horizontal cells and amacrine cells, and increase the gain of the light response in photoreceptors. However, many of the mechanisms controlling the production of NO and cGMP remain unclear. Previous studies have shown activation of NO/cGMP production in response to stimulation with N-methyl-d-aspartate (NMDA) or nicotine, and the differential modulation of cGMP production by GABA(A) and GABA(C) receptors (GABA(A)Rs and GABA(C)Rs). This study used cGMP immunocytochemistry and NO imaging to investigate how the inhibitory GABAergic and glycinergic systems modulate the production of NO and cGMP. Our data show that blocking glycine receptors (GLYR) with strychnine (STRY) produced moderate increases in cGMP-like immunoreactivity (cGMP-LI) in select types of amacrine and bipolar cells, and strong increases in NO-induced fluorescence (NO-IF). TPMPA, a selective GABACR antagonist, greatly reduced the increases in cGMP-LI stimulated by STRY, but did not influence the increase in NO-IF stimulated by STRY. Bicuculline (BIC), a GABA(A)R antagonist, however, enhanced the increases in both the cGMP-LI and NO-IF stimulated by STRY. CNQX, a selective antagonist for alpha-Amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid hydrobromide/kainic acid (AMPA/KA) receptors, eliminated both the increases in cGMP-LI and NO-IF stimulated by STRY, while MK801, a selective antagonist for NMDA receptors, slightly increased the cGMP-LI and slightly decreased the NO-IF stimulated by STRY. Finally, double labeling of NO-stimulated cGMP and either GLY or GABA indicated that cGMP predominantly colocalized with GLY. Taken together, these findings support the hypothesis that GLY and GABA interact in the regulation of the NO/cGMP signaling pathway, where GLY primarily inhibits NO production and GABA has a greater effect on cGMP production. Such interacting inhibitory pathways could shape the course of signal transduction of the NO/cGMP pathway under different physiological situations.     

Activation of the cGMP/nitric oxide signal transduction system by nicotine in the retina

Acetylcholine is one of the primary excitatory neurotransmitters/neuromodulators in the retina, but little is known about the downstream signaling pathways it can activate. The present study immunocytochemically examines the potential sources of acetylcholine and the location of the nicotinic cholinergic receptors in the turtle retina. It also examines how activation of these receptors can influence the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signal-transduction pathways. Photoreceptors, amacrine cells, and potentially ganglion cells contain choline acetyltransferase-like immunoreactivity (LI). Nicotinic acetylcholine receptors are immunocytochemically localized on photoreceptors, horizontal, bipolar, and ganglion cells. Nitric oxide imaging indicates that stimulation with nicotine increases NO production primarily in photoreceptors, horizontal, Muller, bipolar, and ganglion cells. In turn, very select populations of amacrine cells respond to this NO with increased levels of cGMP-LI. Selective inhibitors reveal that nitric oxide synthase is involved in most, but not all, of these increases in cGMP-LI. These results show that acetylcholine can activate the NO/cGMP signal-transduction pathways in both the inner and outer retina. This indicates that both of the major excitatory retinal transmitters, glutamate and acetylcholine, can stimulate NO production that increases levels of cGMP-LI in overlapping populations of retinal cells.     

Morphine-induced nitric oxide production in isolated, iris-ciliary bodies

Considerable evidence suggests that the nitric oxide (NO)/cGMP signaling pathway plays an integral role in opioid receptor-mediated responses in the cardiovascular and immune systems. Previous studies in our laboratory and others have shown that nitric oxide (NO) plays a role in morphine-induced reduction of intraocular pressure (IOP) and pupil diameter (PD) in the New Zealand white (NZW) rabbit. The present study is designed to determine the effect of morphine on NO production in the isolated, iris-ciliary body (ICB), site of aqueous humor production, as this effect could be associated with morphine-stimulated changes in aqueous humor dynamics and iris function. ICBs obtained from normal NZW rabbits were utilized in these experiments. In some experiments, ICB samples were treated with morphine (1, 10 and 100 μM) for 1 h and later examined for changes in NO levels using a NO detection kit. In other experiments, tissue samples were pretreated with naloxone (non-selective opioid receptor antagonist), L-NAME (non-selective NO-synthase inhibitor) or GSH (sulfhydryl reagent) for 30 min, followed by treatment with morphine (10 μM). Morphine caused a concentration-dependent increase in the release of NO from ICBs. Levels of NO detected in the incubation medium of ICB samples increased from 1.49 ± 0.19 (control) to 8.81 ± 2.20 μM/mg protein (morphine-treated; 100 μM). Morphine-stimulated release of NO was significantly inhibited in tissues pretreated with 10 μM naloxone, L-NAME, or GSH. Results obtained from this study suggest that morphine stimulates NO release from the ICB through a mechanism that involves activation of NO-releasing opioid receptors. These results support the in vivo effects of morphine demonstrated in previous studies.     

Correlations between neuronal nitric oxide synthase and muscarinic M3/M1 receptors in the rat retina

This study determined the different signal pathways involved in M1/M3 muscarinic acetylcholine receptor (mAChR) dependent stimulation of nitric oxide synthase (NOS) activity/cyclic GMP (cGMP) production and nNOS mRNA expression in rat retina. Exposure of the retina to different concentrations of carbachol caused an increase in NOS activity, cGMP production and phosphoinositol (PI) accumulation. The increase in NOS activity and cGMP content was blocked by L-NMMA and ODQ, respectively. Also, phospholipase C (PLC) and calcium/calmodulin (CaM) inhibition prevented the carbachol activation on NOS/cGMP pathways. Both, 4-DAMP and pirenzepine but not AF-DX 116 blocked the increase in NOS and cGMP induced by carbachol. Carbachol-stimulation of M1/M3 mAChR increased nNOS-mRNA levels associated with an increase of endogenous NO and cGMP production. The mechanism appears to occur secondarily to stimulation of PIs turnover via PLC. This triggers a cascade reaction involving CaM and soluble guanylate cyclase leading to NO and cGMP accumulation, that in turn, up regulates nNOS-mRNA gene expression. These results give novel insight into the mechanism involved in the regulation of nNOS-mRNA levels by mAChR activation of retina.     

Effect of VEGF on retinal microvascular endothelial hydraulic conductivity: the role of NO

PURPOSE: Vascular endothelial growth factor (VEGF) increases microvascular permeability in vivo and has been hypothesized to play a role in plasma leakage in diabetic retinopathy. Few controlled studies have been conducted to determine the mechanism underlying the effect of VEGF on transport properties (e.g., hydraulic conductivity [Lp]). This study was conducted to determine the effect of VEGF on bovine retinal microvascular endothelial LP and the role of nitric oxide (NO) and the guanylate cyclase/guanosine 3', 5'-cyclic monophosphate/protein kinase G (GC/cGMP/PKG) pathway downstream of NO in mediating the VEGF response. METHODS: Bovine retinal microvascular endothelial cells (BRECs) were grown on porous polycarbonate filters, and water flux across BREC monolayers in response to a pressure differential was measured to determine endothelial LP RESULTS: VEGF (100 ng/ml) increased endothelial LP: within 30 minutes of addition and by 13.8-fold at the end of 3 hours of exposure. VEGF stimulated endothelial monolayers to release NO and incubation of the BRECs with the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine (L-NMMA; 100 microM) significantly attenuated the VEGF-induced LP increase. It was observed that incubation of the monolayers with the GC inhibitor LY-83583 (10 microM) did not alter the VEGF-mediated LP: response. Addition of the cGMP analogue 8-br-cGMP (1 mM) did not change the baseline LP over 4 hours. Also, the PKG inhibitor KT5823 (1 microM) did not inhibit the response of BREC LP to VEGF. CONCLUSIONS: These experiments indicate that VEGF elevates hydraulic conductivity in BRECs through a signaling mechanism that involves NO but not the GC/cGMP/PKG pathway.     

GABA(A) and GABA(C) receptor antagonists increase retinal cyclic GMP levels through nitric oxide synthase

The nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signal transduction pathway plays a role in every retinal cell type. Previous studies have shown that excitatory glutamatergic synaptic pathways can increase cGMP-like immunoreactivity (cGMP-LI) in retina through stimulation of NO production, but little is known about the role of synaptic inhibition in the modulation of cGMP-LI. Gamma-amino-n-butyric acid (GABA) plays critical roles in modulating excitatory synaptic pathways in the retina. Therefore, we used GABA receptor antagonists to explore the role of GABAergic inhibitory synaptic pathways on the modulation of the NO/cGMP signal-transduction system. Cyclic GMP immunocytochemistry was used to investigate the effects of the GABA receptor antagonists bicuculline, picrotoxin, and (1,2,5,6-tetrahyropyridin-4-yl) methylphosphinic acid (TPMPA) on levels of cGMP-LI. Cyclic GMP-LI was strongly increased in response to the GABA(A) receptor antagonist bicuculline, while the GABA(C) receptor antagonist TPMPA had little effect on cGMP-LI. The GABA(A)/GABA(C) receptor antagonist, picrotoxin, caused a moderate increase in cGMP-LI, which was mimicked by the combination of bicuculline and TPMPA. The nitric oxide synthase inhibitor, S-methyl-L-thiocitrulline (SMTC), blocked the increased cGMP-LI in response to stimulation with either bicuculline or picrotoxin. Treatments with either of the glutamate receptor antagonists (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) partially blocked the increases in cGMP-LI seen in response to bicuculline, but a combination of MK-801 and CNQX completely eliminated these increases. These results suggest that inhibitory synaptic pathways involving both types of GABA receptors work through excitatory glutamatergic receptors to regulate the NO/cGMP signal-transduction pathway in retina.     

Cyclic 3′, 5′-Guanosine Monophosphate Synthesis Induced by Atrial Natriuretic Peptide,C-Type Natriuretic Peptide,and Nitric Oxide in the Rat Retina

This study was undertaken to determine whether pathways exist in the rat retina for atrial natriuretic peptide (ANP)-, C-type natriuretic peptide (CNP)-, and nitric oxide (NO)- cyclic 3′, 5′-guanosine monophosphate (cGMP). Exposure of the retina to ANP (10⁻⁷ mol/L), CNP (10⁻⁷ mol/L), S-nitroso-N-acetylpenicillamine (10⁻⁵ mol/L, SNAP; a NO donor), A23187 (10⁻⁵ mol/L; a Ca²⁺ ionophore), and carbachol (10⁻³ mol/L) caused 1.45 ∼ 1.67-fold increases in cGMP content (P < .01). The increase in cGMP content induced by A23187 was blocked by 2-4-carboxyphenyl · 4455-tetramethyl imidazoline 1-oxyl 3-oxide (10⁻³ mol/L, carboxy-PTIO; a NO scavenger). Both carboxy-PTIO (10⁻³ mol/L) and N^G-nitro-l-arginine (10⁻³ mol/L, l-NNA; a NO synthase inhibitor) blocked the increase in cGMP content induced by carbachol. Atropine (10⁻⁵ mol/L; a muscarinic receptor antagonist) also blocked the cGMP increase induced by carbachol. These data demonstrate that ANP-, CNP-, and NO-cGMP pathways exist in the rat retina and that the NO-cGMP pathway may be linked to the activation of the muscarinic receptor.     

一氧化氮与视网膜缺血-再灌注损伤

一氧化氮（NO）在视网膜缺血-再灌注损伤中占重要地位。视网膜缺血-再灌注时,一氧化氮合成酶（NOS）被多种炎性介质和细胞因子激活,使NO大量生成。NO是一种活性很强的自由基,具有广泛的生物学活性。在缺血-再灌注早期,少量NO可降低缺血缺氧对视网膜的损伤程度;晚期过多的NO可通过多种途径对视网膜造成损害。就目前有关NO在视网膜缺血-再灌注损伤中的研究进展进行综述。     

Dilation of retinal arterioles in response to lactate: role of nitric oxide, guanylyl cyclase, and ATP-sensitive potassium channels

PURPOSE: Lactate, a key metabolite in the retinal tissue, has been implicated in regulating retinal blood flow to match retinal metabolic demand. However, the direct effect of lactate on retinal vascular tone and the possible underlying signaling mechanisms remain unknown. In the present study, the roles of endothelium-derived vasodilators, guanylyl cyclase, and potassium channels were examined in lactate-induced dilation of retinal arterioles in vitro. METHODS: Porcine second-order retinal arterioles were isolated, cannulated, and pressurized to 55 cm H2O lumenal pressure without flow. Diameter changes in response to agonists were recorded with videomicroscopic techniques. RESULTS: All vessels developed basal tone (approximately 70 microm in internal diameter) and dilated dose dependently in response to neutralized L-lactate (0.01-10 mM). Inhibition of cyclooxygenase by indomethacin only slightly reduced the vasodilatory response to lactate. In contrast, blockade of monocarboxylate transporters, nitric oxide (NO) synthase, soluble guanylyl cyclase, and ATP-sensitive potassium (KATP) channels nearly abolished lactate-induced vasodilation. The cGMP phosphodiesterase inhibitor zaprinast enhanced the vasodilation response to lactate. Similar to the lactate-induced response, the vasodilation elicited by S-nitroso-N-acetylpenicillamine, an NO donor that activates cGMP signaling, was also inhibited by the soluble guanylyl cyclase and KATP channel blockers. CONCLUSIONS: These data suggest that uptake of lactate by vascular cells via monocarboxylate transporters causes retinal arteriolar dilation predominantly via stimulation of NO synthase and subsequent activation of guanylyl cyclase. The guanylyl cyclase/cGMP signaling triggers opening of KATP channels for vasodilation. A better understanding of the fundamental signaling pathways responsible for lactate-induced dilation of retinal arterioles may help shed light on the possible mechanisms contributing to the metabolic regulation of retinal blood flow under physiological and pathophysiological conditions.     

Nitric oxide amplifies the rat electroretinogram

It is well established that nitric oxide (NO) participates in retinal signal processing through stimulation of its receptor enzyme, soluble guanylyl cyclase (sGC). However, under pathological conditions such as uveoretinitis, diabetic or ischemic retinopathy, elevated NO concentrations may cause protein S-nitrosation and peroxynitrite formation in the retina, promoting cellular injury and apoptosis. Previous electroretinogram (ERG) studies demonstrated deleterious effects of NO on the retinal light response, but showed no evidence for a role in normal signal processing. To better understand the function of NO in ocular physiology, we investigated the effects of exogenous NO, produced by NO donors with different release kinetics, on the flash ERG of the rat. Within a limited concentration range, NO strongly amplified ERG a- and b-waves, oscillatory potentials, and the scotopic threshold response. Amplification exceeded 100% under dark adaptation, whereas the photopic ERG and the isolated cone response were increased by less than 50%. Blocking photoreceptor-bipolar cell synapses by AP-4 demonstrated a significant increase of the isolated a-wave by NO, and modeling the ERG generator PIII supported photoreceptors as primary NO targets. The sGC inhibitors ODQ and NS2028 did not reduce NO-dependent ERG amplification, ruling out an involvement of the classical NO effector cyclic GMP. Using immunohistochemistry, we show that illumination and exogenous NO altered the S-nitrosation level of the photoreceptor layer, suggesting that direct protein modifications caused by elevated levels of NO may be responsible for the observed phenomenon.     

Effects of blue honeysuckle (Lonicera caerulea L.) extract on lipopolysaccharide-induced inflammation in vitro and in vivo

The aim of the present study was to investigate the effects of blue honeysuckle extract (BHE), which contains high level of phenolic compounds, on endotoxin-induced uveitis (EIU). Male Lewis rats were randomly divided into 5 groups with 14 rats in each (eight rats for collection of aqueous humor, six rats for histologic examination). EIU was induced by a footpad injection of lipopolysaccharide (LPS). 1, 10, or 100 mg of BHE was injected intravenously immediately after LPS injection. The aqueous humor was collected at 24 h after LPS injection, the number of infiltrating cells, protein concentration, nitric oxide (NO), tumor necrosis factor (TNF)-alpha, and prostaglandin (PG)-E2 levels in the aqueous humor were determined. Some eyes were enucleated for histologic examination and immunohistochemical analysis. Immunohistochemical staining with a monoclonal antibody against activated nuclear factor (NF)-kappaB was performed to evaluate the effect of BHE on NF-kappaB activation. To further clarify the anti-inflammatory effect, RAW264.7 cells (a mouse macrophage cell line) were stimulated with LPS in the presence or absence of BHE and its major phenolics, cyanidin 3-glucoside (C3G), cyanidin 3-rutinoside (C3R), chlorogenic acid (CA). Expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) were analyzed by Western blot method. BHE treatment significantly reduced the inflammatory cell infiltration, the protein concentration, the levels of NO, TNF-alpha and PGE2 in the aqueous humor and improved histologic status of the ocular tissue. The number of activated NF-kappaB-positive cells was lower in the iris-ciliary body treated with BHE at 3 h after LPS injection. BHE significantly suppressed the production of NO, PGE2 and TNF-alpha in the culture medium as well as the expression of iNOS and COX-2 by LPS-stimulated RAW264.7 cells in a dose-dependent fashion. C3G, C3R and CA showed no or weak inhibitory effects on the level of inflammatory mediators and the expression of iNOS and COX-2. These results suggest that BHE attenuates the degree of inflammation in eyes with EIU by inhibiting the NF-kappaB dependent signaling pathway and the subsequent production of proinflammatory mediators.     

Sildenafil (Viagra) evokes retinal arteriolar dilation: dual pathways via NOS activation and phosphodiesterase inhibition

PURPOSE: Sildenafil (Viagra; Pfizer, New York, NY), a selective phosphodiesterase type-5 (PDE5) inhibitor, is widely used to treat impotence by improving penile blood flow via elevation of cGMP. However, its effect on ocular circulation is controversial and whether retinal arterioles are responsive to this drug remains unclear. In this study, the direct reaction of retinal arterioles to sildenafil was examined and the signaling pathway underlying this vasomotor activity was probed. METHODS: Retinal arterioles from porcine eyes were isolated, cannulated, and pressurized without flow. Diameter changes in response to sildenafil were recorded using videomicroscopic techniques. RESULTS: Retinal arterioles (67 +/- 2 microm) dilated dose dependently to sildenafil (1 ng/mL to 1 microg/mL). This dilation was inhibited by the nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME), the guanylyl cyclase inhibitor 1H- [1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), the extracellular signal-regulated kinase (ERK) pathway inhibitor PD98059, the nonselective potassium channel blocker tetraethylammonium (TEA), and the selective adenosine triphosphate (ATP)-sensitive potassium (K(ATP)) channel blocker glibenclamide. The vasodilation elicited by the NO donor S-nitroso-N-acetylpenicillamine (SNAP) was inhibited by ODQ and TEA but was insensitive to PD98059. In the presence of L-NAME, the addition of SNAP (1 microM) produced modest vasodilation and the inhibited sildenafil response was subsequently restored. The restored dilation was insensitive to PD98059 but was blocked by TEA. CONCLUSIONS: Activation of NO synthase, through ERK signaling, leading to NO production and subsequent guanylyl cyclase activation and K(ATP) channel opening is the major vasodilatory pathway for sildenafil in retinal arterioles. Moreover, the elevated cGMP, from endogenous or exogenous NO, plays a permissive role for sildenafil to exert vasodilation through inhibition of the PDE5 pathway independent of ERK signaling.     

TGF-BETA信号通路介导的眼组织纤维化在眼病发生发展中作用的研究进展

眼科疾病的发生发展与眼组织的异常发育和功能障碍有关,其中相关信号通路的异常活化可使眼组织纤维化进而使其生理功能发生障碍,在眼病的发生和发展过程中发挥重要作用。转化生长因子β(TGF-β)信号通路广泛存在于各类细胞中,可参与细胞的生长、增殖和分化调节,是介导组织发生纤维化的关键信号通路。有研究发现,TGF-β信号通路可在青光眼、眼眶纤维化、增生性玻璃体视网膜病变等眼病中发挥作用。本文就TGF-β信号通路介导的眼组织纤维化在眼病发生发展中的作用研究进展进行简要综述。     

Ocular hypotensive activity of BOL-303259-X, a nitric oxide donating prostaglandin F2α agonist, in preclinical models

The aim of the study was to investigate the ocular hypotensive activity of a nitric oxide (NO)-donating latanoprost, BOL-303259-X, following topical administration. The effect of BOL-303259-X (also known as NCX 116 and PF-3187207) on intraocular pressure (IOP) was investigated in monkeys with laser-induced ocular hypertension, dogs with naturally-occurring glaucoma and rabbits with saline-induced ocular hypertension. Latanoprost was used as reference drug. NO, downstream effector cGMP, and latanoprost acid were determined in ocular tissues following BOL-303259-X administration as an index of prostaglandin and NO-mediated activities. In primates, a maximum decrease in IOP of 31% and 35% relative to baseline was achieved with BOL-303259-X at doses of 0.036% (9 μg) and 0.12% (36 μg), respectively. In comparison, latanoprost elicited a greater response than vehicle only at 0.1% (30 μg) with a peak effect of 26%. In glaucomatous dogs, IOP decreased from baseline by 44% and 10% following BOL-303259-X (0.036%) and vehicle, respectively. Latanoprost (0.030%) lowered IOP by 27% and vehicle by 9%. Intravitreal injection of hypertonic saline in rabbits increased IOP transiently. Latanoprost did not modulate this response, whereas BOL-303259-X (0.036%) significantly blunted the hypertensive phase. Following BOL-303259-X treatment, latanoprost acid was significantly elevated in rabbit and primate cornea, iris/ciliary body and aqueous humor as was cGMP in aqueous humor. BOL-303259-X lowered IOP more effectively than latanoprost presumably as a consequence of a contribution by NO in addition to its prostaglandin activity. The compound is now in clinical development for the treatment of glaucoma and ocular hypertension.     

NCX 667, a Novel Nitric Oxide Donor,Lowers Intraocular Pressure in Rabbits,Dogs, and Non-Human Primates and Enhances TGFβ2-Induced Outflow in HTM/HSC Constructs

PurposeNCX 667, a novel nitric oxide (NO) donor with an isomannide core, was characterized for its IOP-lowering ability in animal models of ocular hypertension and glaucoma. Bioengineered human trabecular meshwork/Schlemm''s canal (HTM/HSC) constructs were used to explore the mode of action.MethodsOcular normotensive New Zealand white (NZW) rabbits (ONT-rabbits), spontaneously ocular hypertensive pigmented Dutch-belted rabbits (sOHT-rabbits), hypertonic saline (5%)–induced transient ocular hypertensive NZW rabbits (tOHT-rabbits), ocular normotensive Beagle dogs (ONT-dogs), and laser-induced ocular hypertensive cynomolgus monkeys (OHT-monkeys) were used. NCX 667 or vehicle (30 µL) was instilled in a crossover, masked fashion and intraocular pressure (IOP) measured before dosing (baseline) and for several hours thereafter. The ONT-rabbits were used for cyclic guanosine monophosphate (cGMP) determination in ocular tissues after ocular dosing with NCX 667. Transforming growth factor-beta2 (TGFβ2) (2.5 ng/mL, six days)–treated HTM/HSC constructs were used to address changes in outflow facility.ResultsNCX 667 resulted in robust and dose-dependent IOP decrease in all models used. Maximal IOP-lowering efficacy at 1% was −4.1 ± 0.6, −12.2 ± 2.7, −10.5 ± 2.0, −5.3 ± 0.8, and −6.6 ± 1.9 mmHg, respectively, in ONT-dogs, sOHT-rabbits, tOHT-rabbits, ONT-rabbits, and OHT-monkeys. In ONT-rabbits NCX 667 (1%) increased cGMP in aqueous humor (AH) but not in retina and iris/ciliary body. NCX 667 concentration-dependently increased outflow facility in TGFβ2-treated HTM/HSC constructs (outflow facility, 0.10 ± 0.06 and 0.30 ± 0.10 µL/min/mmHg/mm², respectively, in vehicle- and NCX 667–treated constructs).ConclusionsNCX 667 leads to robust IOP lowering in several animal models. Evidence in HTM/HSC constructs indicate that the IOP reduction likely results from NO-mediated increase of the conventional outflow pathway. Other mechanisms including changes in AH production and episcleral vein pressure may not be excluded at this time.