Immunolocalization of prostanoid EP receptor isotypes in human trabecular meshwork.

PURPOSE: To assess the localization of the EP-type prostanoid receptors in the human trabecular meshwork (TM) and to determine their spatial distribution in relation to the contractile a-smooth muscle actin fibres. METHODS: Cryosections of human anterior segments were obtained from 17 different donors and immunostained with different EP receptor subtype specific antibodies. Double staining for the EP2 receptor and smooth muscle actin was carried out. Western blots of TM protein samples were studied. RESULTS: No specific staining for the EP1 receptor was observed. The antibodies against the EP2 receptor revealed in all donors intense staining of human trabecular cells throughout the meshwork. EP3 receptor specific staining was not detected. EP4 immunostaining was confined to the corneoscleral region near Schwalbe's line. On western blots, the EP2 receptor was detected. In the posterior TM, the EP2 receptor staining was associated with the dense network of actin fibres. CONCLUSIONS: These immunocytochemical results present evidence that the EP2 receptor is the most abundantly expressed isotype of the PGE receptors in the human TM. This conclusion is in agreement with our previous findings at the transcript level. The relaxant responses of the TM to application of EP2 receptor agonists, and flow enhancement evoked by prostaglandin PGE1, may be explained by the close spatial association of the EP2 receptor with actin fibres.

Elevation of nitric oxide production in human trabecular meshwork by increased pressure.

BACKGROUND: The presence of the nitric oxide (NO)-producing enzyme nitric oxide synthase (NOS) in the trabecular meshwork and the effect of various drugs acting through the NO pathway on the outflow facility and trabecular contractility suggest a role for NO in the regulation of outflow and intraocular pressure (IOP). METHODS: To model the effect of elevated IOP on the NO production in the trabecular meshwork, we perfused anterior segments of human donor eyes in vitro and studied the effect of raised perfusion pressure on NO levels in the perfusate. Furthermore, we evaluated using quantitative PCR whether enhanced perfusion pressure had an effect on the NOS gene expression levels. RESULTS: Elevating the pressure from 10 mmHg to 25 mmHg caused a significant increase in NO production from 3.6+/-0.9 pmol/min to 5.9+/-1.6 pmol/min (mean +/- SEM), corresponding to an average increase of 66%. These high NO levels were reduced by application of L-NAME, an NOS inhibitor, to 2.6+/-0.6 pmol/min. Addition of L-NAME before raising the pressure decreased the basal NO production but was not able to block the increase in NO production after raising the pressure. The transcript level of iNOS was significantly increased in the trabecular meshwork after raising the perfusion pressure. CONCLUSION: These results show that NO production increased after elevation of the pressure gradient over the trabecular meshwork, accompanied by an upregulation of iNOS gene expression. Previous studies have demonstrated that enhanced NO levels facilitate outflow. Taken together, the data indicate the existence of a regulatory feedback mechanism in the trabecular meshwork, which may contribute to the regulation of the IOP. In this system an increase in IOP will enhance NO production, which, in turn, increases the outflow facility, leading to a normalization of the IOP.

Optineurin gene expression level in human trabecular meshwork does not change in response to pressure elevation.

Mutations in the gene optineurin (OPTN) have been associated with primary-open angle glaucoma. Here we present a study on the level of OPTN gene expression in the human trabecular meshwork in response to increased perfusion pressure in the anterior chamber perfusion model of the human eye. Perfusion pressure was raised from 10 to 30 mm Hg for periods ranging between 1 and 24 h. OPTN transcript levels in the trabecular meshwork were determined using real-time quantitative polymerase chain reaction. The results show no statistically significant alteration of the OPTN transcript level after raising the pressure. Moreover, no changes were detected in the transcript levels of the 3 known OPTN isoforms. This result shows that enhanced pressure levels do not lead to rapid changes in gene expression levels of OPTN in human trabecular meshwork. This suggests that alterations in OPTN gene expression are not involved in the mechanisms regulating aqueous humor outflow after an increase in intraocular eye pressure.

Nitric oxide/guanylate cyclase pathways and flow in anterior segment perfusion.

BACKGROUND: The nitric oxide/guanylate cyclase pathway has been suggested to participate in the regulation of intraocular pressure. In the present study, the involvement of nitric oxide pathways on the outflow through the trabecular meshwork was assessed using pharmacological manipulation of the nitric oxide pathway. METHODS: Anterior segments of human donor eyes were maintained in an organ culture perfusion system, and the effects of L-NAME, an inhibitor of nitric oxide synthase, on the flow rate was determined. In a second series, the effects of consecutive application of L-arginine as substrate for nitric oxide synthase, L-NAME, and sodium nitroprusside, a nitric oxide-donor, were studied. The cyclic GMP levels in the perfusate were assessed with an ELISA immunoassay kit. RESULTS: In the first series of experiments, L-NAME caused a statistically significant decrease in flow rate of 10%, accompanied by a decrease in cGMP levels. In the second series, L-arginine did not alter flow, and the effect of L-NAME seen in the first series was prevented by the high preload of L-arginine. Nitroprusside caused a significant 10% increase of flow rate. In the perfusate, cGMP levels were not altered by L-arginine and L-NAME, but were increased after nitroprusside. CONCLUSION: Under organ culture perfusion conditions, modulation of the nitric oxide/guanylate cyclase system alters the flow rate through the trabecular meshwork within a total range of 20%; i.e. the difference between inhibition of NO synthesis and the presence of a NO-donor. These results indicate that the nitric oxide/guanylate cyclase system plays a role in aqueous humour dynamics and, therefore, in the regulation of intraocular pressure.

Effects of elevated pressure on prostanoid receptor gene expression levels in human trabecular meshwork.

The purpose of this study was to assess the effects of increased intraocular pressure on the expression levels of the prostanoid receptor genes (DP, EP(1-4), FP, IP, TP) in the trabecular meshwork of human donor eyes. Anterior segments of human donor eyes were perfused in an ex vivo anterior segment perfusion system under different pressure regimes. The expression levels of the prostanoid receptors and of several housekeeping genes were assessed by non-competitive real-time quantitative polymerase chain reaction (Q-PCR). Perfusion of the anterior segments for 24 h at 10 mm Hg, followed by 24 h at 30 mm Hg, caused a significant decrease in the expression of the EP(2) receptor compared to a constant perfusion under 10 mm Hg. No significant changes were found for the other prostanoid receptor transcripts. When the pressure was raised to 30 mm Hg for only 1 or 3 h, no changes in the EP(2) receptor expression levels were evident. However, a transient DP and TP receptor expression increase was found after 1-hour perfusion at 30 mm Hg. Whether these changes in expression are part of a response of the trabecular meshwork cells in order to normalise intraocular pressure needs to be studied further.