Biomedical subjects

Thomas Yorio

Publications and source records attributed to Thomas Yorio.

18 recordsLinked to original sources

PubMed2006

Dexamethasone inhibition of trabecular meshwork cell phagocytosis and its modulation by glucocorticoid receptor beta.

Glucocorticoid treatment can lead to the development of glaucomatous ocular hypertension and a secondary open-angle glaucoma due to increased aqueous humor outflow resistance that is associated with morphological and biochemical changes in the trabecular meshwork (TM). The cellular responses of glucocorticoids are achieved by binding to the glucocorticoid receptor alpha (GRalpha), a ligand-activated transcription factor. An alternatively spliced variant, glucocorticoid receptor beta (GRbeta), has dominant negative activity on GRalpha and has been implicated in a variety of steroid-resistant diseases. We previously showed that GRbeta can block dexamethasone (DEX) responsiveness in TM cells. TM cells are actively phagocytic and function in the removal of debris, pigment and other materials from the aqueous outflow drainage pathway. A decrease in phagocytic activity has been proposed in the pathogenesis of glaucoma and glucocorticoid-induced glaucoma. In this study, we investigated the effect of DEX and GRbeta on phagocytosis in normal and glaucomatous TM cells. Human transformed normal NTM-5 and primary normal NTM174-00 cells, which express relatively high amounts of GRbeta, and transformed glaucomatous GTM-3 and primary glaucomatous GTM520-05 cells, which have lower GRbeta expression, were treated with 100 nM DEX or vehicle control for 24h. NTM cells also were transfected with a control or GRbeta expression plasmid to examine the effect of GRbeta on phagocytic activity. The cells were incubated with Alexa 488 conjugated Staphylococcus aureus bioparticles opsonized with rabbit IgG for 1h, followed by fixation and incubation with Alexa 633 conjugated goat anti-rabbit IgG to distinguish ingested from extracellular bioparticles. DAPI nuclear staining was used to quantify cell numbers. Cells and bioparticles were visualized by confocal microscopy. We found that NTM-5 cells ingested more bioparticles than GTM-3 cells. DEX treatment significantly decreased the phagocytosis of bioparticles in NTM-5 and GTM-3 cells, while GTM-3 cells were more responsive to DEX, compared to NTM-5 cells. In primary cell culture, NTM174-00 also engulfed more bioparticles than GTM520-05 cells. DEX treatment significantly decreased the phagocytic activity in GTM520-05, but not in NTM174-00 cells. Transient transfection of pCMX-hGRbeta plasmid increased the expression of GRbeta and consequently maintained the phagocytotic activity of NTM-5 cells in the presence of DEX. Our data demonstrated that the expression level of GRbeta in TM cells can regulate DEX-induced suppression of phagocytotic activity. The lower expression of GRbeta in glaucomatous TM cells may contribute to the altered phagocytic function of TM cells, and may lead to the increased aqueous humor outflow resistance mediated by glucocorticoids.

Anti-Inflammatory Agents↗

PubMed2006

Heat shock protein 90 is an essential molecular chaperone for nuclear transport of glucocorticoid receptor beta.

PURPOSE: Glucocorticoid sensitivity in glaucoma has been attributed to differences in the expression of the two glucocorticoid receptors, GRalpha and GRbeta. GRalpha undergoes steroid-dependent nuclear translocation by associating with a heat shock protein (Hsp)90 multiprotein heterocomplex. The nuclear transport of the non-ligand-binding GRbeta is still unknown. In this study, the roles of Hsp90 in the nuclear transport of GRbeta were investigated. METHODS: Immunocytochemistry and Western blot analysis were performed to detect the subcellular expression of GRbeta and Hsp90 in normal and glaucomatous trabecular meshwork (TM) cells, as well as in TM cells overexpressing GRbeta. The role of Hsp90 in GRbeta transport and stability were determined with the Hsp90 inhibitor, 17-AAG and the proteasome inhibitor lactacystin. Coimmunoprecipitation was performed to study GRbeta-Hsp90 complexes. RESULTS: In normal and glaucomatous TM cells, the nuclear concentration of Hsp90 correlates with the nuclear expression of GRbeta. Transfection with a GRbeta expression construct produces an overexpression and accumulation of GRbeta in the nucleus with a corresponding increase in nuclear Hsp90 amount. 17-AAG, a specific Hsp90 inhibitor, completely blocks the nuclear accumulation of GRbeta and consequently leads to the degradation of GRbeta in proteasomes. Coimmunoprecipitation experiments verify that GRbeta complexes with Hsp90 and the microtubule motor protein dynein. CONCLUSIONS: These data provide strong evidence that Hsp90 is an essential molecular chaperone for the nuclear transport of GRbeta. This transport appears to occur along micotubular tracks. Because nuclear GRbeta is important in regulating the glucocorticoid responsiveness, changes in GRbeta nuclear transport could influence subsequent responses that are often seen clinically, such as glucocorticoid resistance in some inflammatory and autoimmune diseases or enhanced glucocorticoid sensitivity in glaucoma.

Acetylcysteine↗

PubMed2005

Endothelin-1 modulates anterograde fast axonal transport in the central nervous system.

Anterograde fast axonal transport (FAxT) maintains synaptic function and provides materials necessary for neuronal survival. Localized changes in FAxT are associated with a variety of central nervous system (CNS) neuropathies, where they may contribute to inappropriate remodeling, a process more appropriately involved in synaptic plasticity and development. In some cases, developmental remodeling is regulated by localized secretion of endothelins (ETs), neuroinflammatory peptides that are also pathologically elevated in cases of neurologic disease, CNS injury, or ischemia. To investigate the potential role of ETs in these processes, we decided to test whether locally elevated endothelin-1 (ET-1) modulates FAxT in adult CNS tissues. We used the established in vivo rat optic nerve model and a novel ex vivo rat hippocampal slice model to test this hypothesis. In vivo, exogenously elevated vitreal ET-1 significantly affected protein composition of FAxT-cargos as well as the abundance and peak delivery times for metabolically-labeled proteins that were transported into the optic nerve. Proteins with molecular weights of 139, 118, 89, 80, 64, 59, 51, 45, 42, 37, and 25 kDa were evaluated at injection-sacrifice intervals (ISIs) of 24, 28, 32, and 36 hr. In acute hippocampal slices maintained on nonvascular supplies of glucose and oxygen, ET-1 significantly decreased the distance traveled along the Schaffer collateral tract by nonmetabolically-labeled lipid rafts at 5 and 10 min after pulse-labeling. In both models, ET-1 significantly affected transport or targeted delivery of FaxT-cargos, suggesting that ET-1 has the potential to modulate FAxT in adult CNS tissues.

Animals↗

PubMed2005

Effect of elevated intraocular pressure on endothelin-1 in a rat model of glaucoma.

The role of endothelin-1 (ET-1) a potent vasoactive peptide, in glaucoma pathogenesis is receiving increasing attention, particularly in astroglial activation in optic nerve damage. Our laboratory has also shown that ET-1 treatment causes proliferation of cultured human optic nerve head astrocytes to possibly initiate astrogliosis. ET-1 is distributed in retina, optic nerve, and ciliary epithelium, however the effects of elevated intraocular pressure (IOP) (as seen in glaucoma) on ET-1 and ET(B) receptors are not clearly understood. In the present study, the levels of immunoreactive ET-1 (ir-ET-1) in aqueous humour (AH) and optic nerve head (ONH) were determined in the Morrison elevated IOP model of glaucoma. Additionally in the ONH of these rats, immunohistochemical analyses of ET(B) receptors and glial fibrillary acidic protein (GFAP; a marker for astroglial cells and for astrogliosis) were performed. There was 2- to 2.5-fold increase in AH ir-ET-1 levels for rats subjected to elevated IOP, compared to their respective controls. In the Morrison rat model of glaucoma, elevated IOP increased optic nerve ir-ET-1 with concomitant increases in ir-ET(B) and ir-GFAP labelling (possibly indicative of astrogliosis and hypertrophy). As seen in brain astrocytes subjected to neurotrauma, the present findings are suggestive of ET-1's role in astroglial activation, particularly in response to elevated IOP in glaucoma.

Animals↗

PubMed2005

Characterization of endothelin-converting enzyme activities in ARPE-19 cells, a human retinal pigmented epithelial cell line.

Elevated endothelin-1 (ET-1) levels are detected in patients with glaucoma. ET-1 is produced from its precursor, Big ET-1, by endothelin-converting enzyme (ECE). Characterization of ET- 1 secretion and ECE activity was performed in ARPE-19 cells, a human retinal pigmented epithelial cell-line. The ET(B) receptor but not the ET(A) receptor was detected by immunoblotting and cross-linking using 125I-ET-1 at the plasma membrane (PM). Tumor necrosis factor-alpha (10 nmol/L) induced a 700% increase in ET-1 levels and such an effect was further potentiated by BQ788, an ET(B) receptor antagonist, suggesting the involvement of ET(B) receptor in ET-1 clearance. Big ET-1-converting activities were detected in both the PM and cytosol. Phosphoramidon, thiorphan, acidification, and phenanthroline inhibited PM ECE activity; the cytosolic ECE activity was not affected by phenanthroline but was inhibited by the others. In contrast, ECE cytosolic activities were activated by acidification (pH 6.4), suggesting the involvement of ECE-2 or cathepsin-like activity. Pepstatin, a potent inhibitor of cathepsins, and phosphoramidon, a potent inhibitor of ECE, inhibited the cytosolic conversion of Big ET-1 peptide by 46% and 35%, respectively, whereas the combination of both inhibited the cytosolic activity by 93%. Based on immunoblotting, ECE-1 was detected only at the PM, whereas ECE-2 and cathpesins B and D were detected in the cytosol. In summary, ET-1 production in RPE is regulated by at least two isoforms of ECE, (cytosolic and PM) as well as cathepsins.

Aspartic Acid Endopeptidases↗

PubMed2005

Localization of endothelin-converting enzyme in bovine optic nerve and retina.

A significant loss and remodeling of the lamina cribrosa tissue leading to the excavation of the optic nerve is seen in glaucoma. Elevated endothelin-1 (ET-1) levels are detected in the aqueous humor of patients of open-angle glaucoma and in the plasma of patients with normal- tension glaucoma. Optic nerve damage, including axonal loss, can be mimicked by ET-1 injection near the optic nerve. ET-1 is produced from its precursor Big ET-1 (38 amino acids) by endothelin-converting enzyme (ECE). Although ET-1 and its receptors have been identified in the retina, little is known of the distribution of ECE at the optic nerve. Presently, ET-1 receptors and Big ET-1 converting activities were characterized in bovine optic nerve and the retina. The ET(B) receptor was detected in both the optic nerve and retina by immunoblotting and cross-linking, using 125I-ET-1. However, the ET(A) receptor was detected only in the retina. Big ET-1 conversion activities were detected in the plasma membrane (PM) of bovine retina, but not in the PM of the optic nerve. The retinal PM Big ET-1 converting activity was inhibited by phosphoramidon, thiorphan, and acidification. Furthermore, ECE cytosolic activities were detected in both the optic nerve and retina. Unlike the PM-ECE, cytosolic Big ET-1 converting activities were activated by acidification (pH 6.4), suggesting the involvement of ECE-2-like activity and/or cathepsin activity. Pepstatin, a potent inhibitor of cathepsins, inhibited the optic nerve (ON) cytosolic conversion of Big ET-1 peptide by 50%, and the combination of pepstatin and phosphoramidon, a potent inhibitor of ECE, inhibited the ON cytosolic activity by 86%. By contrast, the combination of both inhibitors weakly inhibited the cytosolic retinal Big ET-1 converting activity. Western blotting revealed the presence of ECE-1 at the PM of the retina not the ON. ECE-2 and cathpesins B, D, and L were detected only in the cytosol of both the retina and ON. In summary, it appears that ET-1 could be produced in the retina and optic nerve by at least two ECE subtypes and, perhaps, cathepsins. Big ET-1 converting activity may be an important target in preventing ET-1-induced optic nerve pathology.

Animals↗

PubMed2005

Regulation of glucocorticoid responsiveness in glaucomatous trabecular meshwork cells by glucocorticoid receptor-beta.

PURPOSE: Glucocorticoid administration can lead to increased intraocular pressure in greater than 90% of patients with primary open-angle glaucoma (POAG), compared with 30% to 40% of the general population. The molecular mechanisms for increased steroid responsiveness among patients with glaucoma are unknown. An alternative splicing variant of the human glucocorticoid receptor GRbeta has dominant negative activity and has been implicated in a variety of steroid-resistant diseases. GRbeta also may play a role in glucocorticoid hyperresponsiveness in glaucoma. METHODS: Western blot analysis was performed to detect the expression of GRalpha and GRbeta in TM cells and its regulation by dexamethasone (DEX). Immunocytochemistry was used to compare the subcellular expression of GRbeta between normal and glaucomatous TM cell lines. DEX transgene induction in a luciferase reporter was performed to investigate the differential glucocorticoid responsiveness between multiple normal and glaucomatous TM cell lines. Overexpression of GRbeta was conducted in glaucomatous TM cell lines, and the regulation of GRbeta in the Dex-induced reporter gene luciferase or endogenous myocilin and fibronectin expression were determined. RESULTS: Trabecular meshwork (TM) cell lines derived from normal individuals expressed higher levels of GRbeta than did glaucomatous TM cells. Glaucomatous TM cells were more susceptible to DEX induction of a luciferase reporter gene than were TM cells derived from normal donors. Overexpression of GRbeta in glaucomatous TM cells inhibited DEX induction of a luciferase reporter gene as well as the endogenous genes MYOC and fibronectin. CONCLUSIONS: The decreased amount of GRbeta in glaucomatous TM cells could result in enhanced glucocorticoid responsiveness and ocular hypertension.

Adolescent↗

PubMed2004

Hypoxia augments TNF-alpha-mediated endothelin-1 release and cell proliferation in human optic nerve head astrocytes.

The effect of hypoxia (24 h) on TNF-alpha-mediated release of endothelin-1 (ET-1) from human optic nerve head astrocytes (hONAs) and TNF-alpha- and ET-1-induced hONA proliferation was determined. ET-1 synthesis and release was quantitated using ELISA while TNF-alpha (10 nM)- and ET-1 (100 nM)-mediated hONA proliferation was assessed by CellTiter 96 aqueous one-solution cell proliferation assay, respectively. hONAs appeared to be more rounded with fewer processes following 24 h hypoxia compared to thodr seen in normoxia. Hypoxia enhanced TNF-alpha-mediated ET-1 synthesis and release (by 5-fold) and also significantly increased TNF-alpha- and ET-1-mediated hONA proliferation. PD142893 (1 microM), an ET(A/B) receptor antagonist, blocked ET-1-mediated hONA proliferation both under normoxia and hypoxia, while doing so only under normoxia following TNF-alpha treatment. Also, U0126 (10 microM; an upstream ERK1/2 inhibitor) completely blocked agonist-induced hONA proliferation in normoxia and partially blocked the same in hypoxia. These results demonstrate for the first time that hONAs secrete ET-1 and that TNF-alpha and hypoxia can regulate its levels. Moreover, hypoxia augments the proliferative responses of hONAs to TNF-alpha and ET-1. These agonist-mediated effects following hypoxia could contribute to astroglial activation as seen in glaucomatous optic nerve heads.

Aged↗

PubMed2004

Endothelin-1 distribution and basolateral secretion in the retinal pigment epithelium.

Endothelins are a family of conserved vasoactive peptides that are widely expressed in different biological systems including the eye. In the cell culture model of retinal pigment epithelium, ARPE-19, the synthesis and secretion of endothelin-1 (ET-1) is regulated by cholinergics and TNF-alpha. In the present study we investigated the expression of ET-1 in RPE in situ, in rat and human eyes. Additionally, we have employed the human retinal pigment epithelial (ARPE-19) cells to delineate the apical and basolateral ET-1 expression and secretion by confocal microscopy and radioimmunoassay, respectively. Our results suggest a possible conservation of ET-1 expression predominantly in the mammalian RPE underlining its importance at this site. Additionally, our results suggest that constitutive ET-1 secretion is predominantly towards the basolateral side in cultured RPE possibly allowing ET-1 to activate its receptors located in the choroidal blood vessels and regulate retinal and choroidal blood flow.

Animals↗

PubMed2003

Dexamethasone regulates endothelin-1 and endothelin receptors in human non-pigmented ciliary epithelial (HNPE) cells.

Endothelin-1 (ET-1) lowers intraocular pressure (IOP) in animal models by regulating aqueous humour dynamics through both inflow and outflow mechanisms. Moreover, ET's concentration is elevated in glaucoma patients and in animal models of glaucoma. Glucocorticoid therapy often can lead to increase IOP in susceptible individuals including patients with primary open angle glaucoma (POAG). In this study, we examined the effects of dexamethasone (Dex), a frequently used anti-inflammatory glucocorticoid, on the synthesis and release of endothelin-1 and on the expression of endothelin receptors in human non-pigmented ciliary epithelial (HNPE) cells, an established source for ET-1 in the anterior chamber. As measured by ET-1 immunoreactivity, ET-1 was concentration-dependently increased following 24hr Dex treatment, with a maximum concentration (100 nM) causing a threefold increase of ET-1 release. Western blot analysis of HNPE cells showed the expression of endothelin receptor A (ET(A)) and endothelin receptor B (ET(B)) with approximate molecular weights of 40 kDa. Dex treatment decreased ET(A) receptor expression at all Dex doses, but up-regulated ET(B) receptors with 10nM Dex having the greatest effect. Quantitative PCR demonstrated that Dex also increased the mRNA of pre-pro-ET-1 (ppET-1) and ET(B) but decreased the mRNA of ET(A). RU486, a glucocorticoid receptor antagonist, was able to block Dex's actions on ET release and ET(B) receptor expression, but did not block its action on ET(A) receptor expression. Endothelin receptors were minimally expressed in HNPE cells as determined in binding experiments (B(max): ET(A) 17, ET(B) 25 fmolmg(-1) membrane protein). However Dex treatment stimulated a dramatic increase in ET(B) receptor density while decreasing ET(A) receptors (B(max): ET(A) 11, ET(B) 116 fmolmg(-1) membrane protein). The regulation of endothelin and its receptors could be a novel mechanism associated with glucocorticoid's effects on intraocular pressure. The increase in ET-1 and disproportionate regulation in ET receptor expression by Dex could promote dysregulation in ET's mechanism on both inflow and outflow, thus affecting aqueous humour dynamics in the anterior chamber of the eye.

Anti-Inflammatory Agents↗

PubMed2003

Eyeing endothelins: a cellular perspective.

Endothelin is an endogenous vasoactive peptide that is considered among the most potent vasoconstrictor substances known. In addition to its vascular effects, endothelins and their receptors have been shown to be present in the eye and to have a number of ocular actions that may be important for ocular homeostasis, but, in excess can be a potential contributor to ocular neuropathy in glaucoma. The current review focuses on the cellular and molecular aspects of endothelins and its receptors in the eye with an emphasis on its relationship to ocular function and its potential role in the etiology of glaucoma pathophysiology.

Animals↗

PubMed2003

Ophthalmic drug discovery.

Millions of people suffer from a wide variety of ocular diseases, many of which lead to irreversible blindness. The leading causes of irreversible blindness in the elderly--age-related macular degeneration and glaucoma--will continue to effect more individuals as the worldwide population continues to age. Although there are therapies for treating glaucoma, as well as ongoing clinical trials of treatments for age-related macular degeneration, there still is a great need for more efficacious treatments that halt or even reverse ocular diseases. The eye has special attributes that allow local drug delivery and non-invasive clinical assessment of disease, but it is also a highly complex and unique organ, which makes understanding disease pathogenesis and ocular drug discovery challenging. As we learn more about the cellular mechanisms involved in age-related macular degeneration and glaucoma, potentially, new drug targets will emerge. This review provides insight into some of the new approaches to therapy.

Animals↗

PubMed2003

Regulation of Na,K-ATPase expression by endothelin-1 in transformed human ciliary non-pigmented epithelial (HNPE) cells.

Endothelin-1 (ET-1) (1-100 nM) decreases the activity of Na,K-ATPase, a key enzyme responsible for aqueous humor formation, in transformed human non-pigmented ciliary epithelial (HNPE) cells. The present study sought to determine if ET-1 alters the expression of the catalytically active alpha subunit of Na,K-ATPase in HNPE cells and identify mechanisms underlying these effects. We report that acute (15 and 30 min) treatment with ET-1 results in an increase in mRNA expression of the alpha 1 subunit of Na,K-ATPase. Similar to ET-1's effects, ouabain (100 microM), a selective inhibitor of Na,K-ATPase, and monensin (10 microM), a sodium ionophore, also increased Na,K-ATPase expression in HNPE cells. The increase in Na,K-ATPase expression by short-term treatment with ouabain and monensin was dependent on their ability to elevate intracellular sodium concentrations. However, acute ET-1 treatment mediated increase in Na,K-ATPase expression was independent of changes in intracellular sodium. A prolonged (24 hr) ET-1 treatment results in an increase in both mRNA and protein levels of the alpha 1 subunit of Na,K-ATPase. These observations suggest that ET-1 could play a homeostatic role in modulating aqueous humor formation by having differential effects on the activity and expression of Na,K-ATPase by the ciliary epithelium in the eye.

Cell Line, Transformed↗

PubMed2003

Endothelin-1 synthesis and secretion in human retinal pigment epithelial cells (ARPE-19): differential regulation by cholinergics and TNF-alpha.

PURPOSE: Endothelin (ET)-1 can produce nerve damage analogous to that in optic neuropathies such as glaucoma. The precise source of endothelin in the posterior segment of the eye remains unclear. The present study was conducted to determine whether the retinal pigment epithelium (RPE), which helps maintain the outer blood-retinal barrier, is a local source for ET-1 and whether the amount of ET-1 secreted by the RPE is be differentially regulated by cholinergics and the cytokine TNF-alpha. METHODS: Human retinal pigment epithelial cells (ARPE-19) were cultured either to a mature state (mRPE) for 4 weeks, with well-defined tight junctions, or to a young state (yRPE) for 4 days, with incompletely formed tight junctions. ET-1-like immunoreactivity was determined by immunocytochemistry, and secreted ET-1 was quantified by radioimmunoassay in both cell types. Cells were stimulated with the cholinergic agonist carbachol or with the cytokine TNF-alpha for specific periods. The expression of muscarinic receptor subtypes M1 and M3 and the peripheral membrane protein ZO-1 were analyzed by immunoblot and immunocytochemistry, respectively. Expression of preproendothelin-1 (ppET-1) mRNA after application of different stimuli at specific time points was determined by real-time RT-PCR. Carbachol-mediated elevation in intracellular calcium ([Ca2+]i) was also measured in the presence or absence of a selective muscarinic receptor antagonist. RESULTS: Constitutive synthesis and secretion of ET-1 was greater in mRPE than in yRPE cells. TNF-alpha caused a significant increase in ppET-1 mRNA levels and ET-1 secretion in both phenotypes. The disruption and subsequent breakdown of the tight junction barrier was evident in either phenotype after treatments with TNF-alpha. There was a concentration-dependent increase in [Ca2+]i in both y- and mRPE cells in response to CCh. CCh at 1 microM significantly increased ET-1 secretion, a response observed in yRPE but not in mRPE cells. This effect may be mediated primarily by the M3 receptor subtype and is phospholipase C (PLC) dependent. CONCLUSIONS: Regulation of ET-1 release in ARPE-19 cells was differentially regulated by TNF-alpha and CCh and was dependent on the age of the culture. RPE may be a source for ET-1 in the retina, and its increased release may become more important during breakdown of the blood-retinal barrier, as seen after TNF-alpha treatment.

Calcium↗

PubMed2003

Interactions of endothelin-1 with dexamethasone in primary cultured human trabecular meshwork cells.

PURPOSE: Concentrations of aqueous humor endothelin (ET)-1 are increased in patients with primary open-angle glaucoma (POAG) as well as in animal models of glaucoma. Glucocorticoids have also been associated with glaucoma, in that topical administration of glucocorticoids can increase intraocular pressure by increasing outflow resistance in the trabecular meshwork (TM) in some individuals. Recent research has shown that dexamethasone (Dex), a synthetic glucocorticoid, can increase the release of ET-1 from human nonpigmented ciliary epithelial (HNPE) cells, a source of aqueous ET-1. In the present study, the downstream interaction of ET-1 with Dex in target TM cells, an action that may alter outflow resistance, was investigated. METHODS: A normal primary human TM (NTM) cell line and a TM cell line derived from a glaucomatous eye (GTM) were used. The cells were treated with vehicle or Dex. The mRNA levels of prepro-ET-1, endothelin receptor A (ET(A)), and endothelin receptor B (ET(B)) were measured by quantitative RT-PCR (QPCR). The protein expression of ET(A) and ET(B) receptors were investigated by Western blot analysis using polyclonal anti-ET(A) and anti-ET(B) antibodies, respectively, on plasma membrane fractions. Intracellular Ca(2+) ([Ca(2+)](i)) mobilization mediated by ET-1 was measured using the Fura-2 AM fluorescent probe technique as an index of ET receptor function. ET-1-stimulated nitric oxide (NO) release was measured using a Griess colorimetric NO synthase assay kit. RESULTS: Both NTM and GTM cultured cells expressed prepro-ET-1 mRNA less abundantly than did HNPE cells, and Dex treatment had no effect on the mRNA expression of the ET-1 gene. TM cells expressed mRNA of ET(A) receptors as detected by QPCR, whereas the ET(B) message was not clearly delineated. Western blot analysis showed that both ET(A) and ET(B) receptor proteins were present. The ET(A) receptor was linked to calcium mobilization as ET-1 produced an increase in intracellular calcium release, and this increase was blocked with a selective ET(A) receptor antagonist. Dex failed to induce any change in the expression of the ET(A) receptor in both NTM and GTM cells, and this was supported by the absence of a Dex effect on the ET-1-induced calcium response. However, Dex treatment diminished ET(B) receptor protein expression and produced a decrease in ET-1-stimulated release of NO, a response mediated by ET(B) receptors in TM cells. CONCLUSIONS: The Dex-induced increase in ET-1 released by HNPE cells coupled to the downstream Dex-induced specific suppression of ET(B) receptor protein expression and declines in ET-1-mediated increase in NO released by TM cells could increase contraction and decrease relaxation of the TM and contribute to the declines in conventional aqueous humor outflow and increases in intraocular pressure that occur with glucocorticoids.

Aged↗

PubMed2002

Endothelin: is it a contributor to glaucoma pathophysiology?

Endothelin is a vasoactive peptide that has been shown to play an important role in vascular homeostasis. Recently, endothelin and its receptors have been found in ocular tissues where it appears to have a regulatory function. Endothelin is found in both the aqueous and vitreous humors and its concentration is elevated in glaucoma patients and in animal models of glaucoma. In the current review, the authors present information about the distribution of endothelin and endothelin receptors in the eye and the ocular actions of endothelins. Specifically, endothelin/aqueous humor dynamics, endothelin/nitric oxide interactions, endothelin and ischemia, and endothelin/optic nerve head effects. Observations concerning the potential role of endothelin in glaucoma pathophysiology is presented and discussed relative to its effects on the optic nerve head and in relation to glaucoma theories.

Aqueous Humor↗

PubMed2002

Human optic nerve head astrocytes as a target for endothelin-1.

PURPOSE: To determine whether human optic nerve head astrocytes (hONAs) are target cells for the actions of endothelin (ET)-1, a potent vasoactive peptide, by causing astrocyte proliferation, as occurs in glaucomatous optic nerve heads. ET-1 levels are elevated in glaucomatous eyes, and administration of ET-1 to the retina causes glial activation and optic nerve damage in animal models in a manner similar to that observed in glaucoma. METHODS: Well-characterized hONAs were used in this study. Cell proliferation of hONAs was assessed, after ET-1 treatment under serum-free culture conditions, with both a formazan assay and [3H]thymidine uptake. ET receptor involvement for cell proliferation was determined with BQ788 (an ETB antagonist), BQ610 (an ETA antagonist), PD142893 (an ET(A/B) mixed antagonist), and sarafotoxin 6C (S6C; an ET(B) agonist). ET-1-induced intracellular calcium ([Ca2+]i) in hONAs was measured by fura-2 imaging. RT-PCR was used to determine whether hONAs express mRNA for preproET-1, ET(A), and ET(B) receptors. RESULTS: ET-1 (10 and 100 nM) caused a time-dependent proliferation of hONAs, which was completely blocked by PD142893, as detected by two different cell proliferation assays. The effects of ET-1 were blocked by BQ788 and were also mimicked by S6C, indicative of the involvement of ET(B) receptor activation. ET-1-induced elevation in [Ca2+]i, and cell proliferation were both blocked completely by the ET(A) antagonist BQ610, suggesting ET(A) receptor involvement. The hONAs expressed mRNA for ET(A) and ET(B) receptors as well as preproET-1, suggesting that these cells may also be a source for ET-1 in the optic nerve head. CONCLUSIONS: ET-1 induces astroglial proliferation in cultured human optic nerve head astrocytes through ET(A/B) receptor activation. This is similar to the proliferation of ET-1 in brain astrocytes. These findings suggest that ET-1, which is elevated in glaucoma, could cause proliferation of ONAs in the optic nerve head.

Aged↗

PubMed2002

Effects of endothelin-1 on components of anterograde axonal transport in optic nerve.

PURPOSE: Increased levels of endothelins (ETs) are associated with glaucoma and have been said to contribute to the development of glaucomatous optic neuropathy. In glaucoma, movement of selected components of anterograde axonal transport essential in ganglion cell survival is impaired-specifically, the transport of mitochondria. This study evaluates the effect(s) of a single administration of intravitreous ET-1 on anterograde axonal transport in the rat optic nerve. METHODS: Proteins for anterograde axonal transport were pulse labeled by intravitreous injection of (35)S-methionine plus or minus ET-1 (2 nmol) in HEPES buffer (pH 7.4). At appropriate time intervals, optic nerves were dissected, sectioned while frozen, and homogenized in denaturing buffer, and transported protein was quantitated by liquid scintillation counting. Counts corrected for efficiency, quench, background, and decay were statistically evaluated (ANOVA, n = 7). RESULTS: Effects of treatment with intravitreous ET-1 on anterograde axonal transport were significant, biphasic, and prolonged (4 hours to 21 days). The initial phase was a significant enhancement of transport at times normally associated with small, fast-moving tubulovesicles (4 and 24 hours), followed by significant impairments at times normally associated with transport of mitochondria (28-36 hours), cytoplasmic matrix (4 days), and cytoskeletal proteins (21 days). The most pronounced effect of ET-1 was decreased axonal transport at times associated with normal anterograde transport of mitochondrial proteins (28, 32, and 36 hours, P = < 0.001, P < 0.015, and P < 0.001, respectively). This was mimicked by ET-3 at 28 hours. CONCLUSIONS: Effects of intravitreous ET-1 are consistent with a receptor-mediated role for elevated ETs in pathologic misregulation(s) of anterograde axonal transport.

Animals↗