Cannabinoid receptors in conjunctival epithelium: identification and functional properties.

PURPOSE: Preservation of the ocular surface barrier requires complex control of epithelial cell proliferation and inflammation mechanisms. The endocannabinoid system may be regulating these processes. Therefore, the authors explored the presence and properties of cannabinoid receptors (CB1 and CB2) in conjunctival epithelial cells. METHODS: The authors used immunohistochemistry to detect CB1 and CB2 in normal mouse conjunctiva, human conjunctival cryosections and impression samples, and IOBA-NHC cells, a human conjunctiva-derived cell line. The presence of CB1 and CB2 proteins and transcripts was studied in IOBA-NHC cells by Western blot and RT-PCR, respectively. The authors also used this cell line to assay cannabinoid ligand-induced changes in cAMP levels, cell growth, and tumor necrosis factor-alpha (TNF-alpha)-induced activation of c-jun N-terminal kinase (JNK) and nuclear factor-kappaB (NF-kappaB). RESULTS: Mouse and human conjunctival epithelial cells displayed CB1 and CB2 proteins and transcripts. Cannabinoid receptor activation decreased cAMP levels in IOBA-NHC cells, and specific CB1 and CB2 antagonists canceled this effect. Cannabinoid ligands also increased cell growth and blocked stress pathways activated by TNF-alpha in vitro. CONCLUSIONS: Cannabinoid receptors are present in mouse and human conjunctival cells. Functional responses, such as decreased cAMP levels, proliferation, and modulation of stress signaling pathways, were mediated by CB1 and CB2 stimulation. Thus, these receptors might be involved in the regulation of epithelial renewal and inflammatory processes at the ocular surface.

Blockade of endothelinergic receptors prevents development of proliferative vitreoretinopathy in mice.

Proliferative vitreoretinopathy (PVR) is characterized by severe glial remodeling. Glial activation and proliferation that occur in brain diseases are modulated by endothelin-1 (ET-1) and its receptor B (ETR-B). Because retinal astrocytes contain ET-1 and express ETR-B, we studied the changes of these molecules in an experimental mouse model of PVR and in human PVR. Both ET-1 and ETR-B immunoreactivities increased in mouse retina after induction of PVR with dispase. Epi- and subretinal outgrowths also displayed these immunoreactivities in both human and experimental PVR. Additionally, myofibroblasts and other membranous cell types showed both ET-1 and ETR-B immunoreactivities. In early stages of experimentally induced PVR, prepro-ET-1 and ETR-B mRNA levels increased in the retina. These mRNA levels also increased after retinal detachment (RD) produced by subretinal injection. Treatment of mice with tezosentan, an antagonist of endothelinergic receptors, reduced the histopathological hallmarks of dispase-induced PVR: retinal folding, epiretinal outgrowth, and gliosis. Our findings in human and in dispase-induced PVR support the involvement of endothelinergic pathways in retinal glial activation and the phenotypic transformations that underlie the growth of membranes in this pathology. Elucidating these pathways further will help to develop pharmacological treatments to prevent PVR. In addition, the presence of ET-1 and ETR-B in human fibrous membranes suggests that similar treatments could be helpful after PVR has been established.

Controlling retinal pigment epithelium injury after experimental detachment of the retina.

PURPOSE: Damage induced by detachment of the neural retina and the retinal pigment epithelium (RPE) can be reduced by dark adaptation. The authors evaluated the influence of the duration of dark adaptation, time of day, and modification of the melatonin-dopamine pathway on acute RPE lesions induced by mechanical detachment. METHODS: BALB/c mice were studied at different times of day and different periods of dark adaptation. Some mice were treated with melatonin or sulpiride, a D2 dopamine receptor antagonist. Enucleated eyes and different saline solutions were used in experiments ex vivo. Retinal detachments in vivo were made by subretinal injections of hyaluronic acid. RPE cell damage was quantitatively evaluated with a dye exclusion procedure, and their viability was tested by preservation of tight junctions in culture. Lectin histochemistry was used to examine the interphotoreceptor matrix (IPM). RESULTS: Significant propidium iodide (PI) incorporation in RPE cells was detected after ex vivo separation during daytime, but it was very low when detachment took place at night after 24 to 48 hours of dark adaptation. PI exclusion was achieved during daytime after a single hour of dark adaptation when mice were pretreated with melatonin or sulpiride. Reduction of RPE cell damage was accompanied by decreased lectin binding to cone sheaths. CONCLUSIONS: A combination of time of day and length of dark adaptation decreased damage induced by detachment of the retina ex vivo and in vivo. Melatonin or sulpiride could replace these environmental factors. Therefore, melatonin and dopamine pathways might be involved in the control of IPM properties and retina/RPE interactions.

Endothelin receptors in light-induced retinal degeneration.

Excessive light exposure leads to retinal degeneration in albino animals and exacerbates the rate of photoreceptor apoptosis in several retinal diseases. In previous studies we have described the presence of endothelin-1 (ET-1) and its receptors (ET-A and ET-B) in different sites of the mouse retina, including the retinal pigment epithelium, the outer plexiform layer (OPL), astrocytes, the ganglion cell layer (GCL), and vascular endothelia. After light-induced degeneration of photoreceptors, endothelinergic structures disappear from the OPL, but ET-1 and ET-B immunoreactivities increase in astrocytes. Here, we present novel observations about the course of light-induced retinal degeneration in BALB-c mice exposed to 1500 lux during 4 days with or without treatment with tezosentan, a mixed endothelinergic antagonist. Retinal whole mounts were immunostained with anticleaved caspase-3 (CC-3) serum to identify apoptotic photoreceptor cells within the outer nuclear layer (ONL). Glial activation was measured as glial fibrillary acidic protein (GFAP) immunoreactivity in retinal whole mounts and in Western blots from retinal extracts. Tezosentan treatment significantly reduced both the number of CC3-immunoreactive cells and GFAP levels, suggesting that inhibition of endothelinergic receptors could play a role in photoreceptor survival. Using confocal double immunofluorescence, we have observed that ET-A seems to be localized in bipolar cell dendrites, whereas ET-B is localized in horizontal cells. Our observations suggest the existence of an endothelinergic mechanism modulating synaptic transmission in the OPL. This mechanism could perhaps explain the effects of tezosentan treatment on photoreceptor survival.

The positive inotropic effect of angiotensin II: role of endothelin-1 and reactive oxygen species.

Many effects believed to be because of angiotensin II (Ang II) are attributable to the action of endothelin (ET)-1, which is released/produced by Ang II. We investigated whether Ang II elicits its positive inotropic effect (PIE) by the action of endogenous ET-1, in addition to the role played by reactive oxygen species (ROS) in this mechanism. Cat cardiomyocytes were used for: (1) sarcomere shortening measurements; (2) ROS measurements by epifluorescence; (3) immunohistochemical staining for preproET-1, BigET-1, and ET-1; and (4) measurement of preproET-1 mRNA by RT-PCR. Cells were exposed to 1 nmol/L Ang II for 15 minutes. This low concentration of Ang II increases sarcomere shortening by 29.2+/-3.7% (P<0.05). This PIE was abrogated by Na+/H+ exchanger or Na+/Ca2+ exchanger reverse mode inhibition. The production of ROS increased in response to Ang II treatment (DeltaROS respect to control: 68+/-15 fluorescence units; P<0.05). The Ang II-induced PIE and ROS production were blocked by the Ang II type 1 receptor blocker losartan, the nonselective ET-1 receptor blocker TAK044, the selective ETA receptor blocker BQ-123, or the ROS scavenger N-(2-mercapto-propionyl)glycine. Exogenous ET-1 (0.4 nmol/L) induced a similar PIE and increase in ROS production to those caused by Ang II. Immunostaining for preproET-1, BigET-1, and ET-1 was positive in cardiomyocytes. The preproET-1 mRNA abundance increased from 100+/-4.6% in control to 241.9+/-39.9% in Ang II-treated cells (P<0.05). We conclude that the PIE after exposure to 1 nmol/L Ang II is due to endogenous ET-1 acting through the ETA receptor and triggering ROS production, Na+/H+ exchanger stimulation, and Na+/Ca2+ exchanger reverse mode activation.

Endothelin-1 and endothelin receptors in light-induced retinal degeneration.

We have studied the distribution of endothelinergic molecules: prepro-endothelin-1 (PPET-1), endothelin-1 (ET-1), and receptors A and B (ET-A) and (ET-B) in the retina of mice. The localization of these molecules in normal mice was compared to their localization in retinas from animals submitted to continuous illumination during 1, 6, 9 or 18 days. We also evaluated the distribution of smooth muscle actin (SMA) and glial markers, glial fibrillary acidic protein (GFAP) and glutamine synthase (GS). PPET-1 immunoreactivity mainly appeared in retinal pigment epithelium (RPE) and cells of the ganglion cell layer (GCL), whereas ET-1 immunoreactivity was present in the RPE, outer plexiform layer (OPL) and astrocytes. Astrocytes exhibited the strongest immunostaining in the retina. ET-A immunoreactivity was observed in endothelium, RPE, OPL and cells of the GCL. By contrast, ET-B immunoreactivity could be detected in endothelial cells, horizontal cells and astrocytes. Astrocytes of the optic nerve also exhibited ET-1, ET-A, and ET-B immunoreactivities. After light-induced degeneration, there was an increase of RPE immunostaining. Degeneration of photoreceptors was accompanied by disappearance of immunoreactivity in the OPL. However, ET-A immunoreactivity appeared in the amacrine sublayer of the INL. There was an enormous increase in astrocytes and its cell processes. The increase of astrocytic immunoreactivities for ET-1 and ET-B was confirmed by quantitative image analysis. Growth of astrocytic cell processes was most marked around retinal blood vessels. Our findings indicate that there are at least three endothelinergic pathways in the normal retina: (1) between the RPE and choriocapillaris, (2) at the OPL, and (3) between blood vessels, astrocytes and cells of the GCL. After light-induced degeneration of photoreceptors, endothelinergic molecules were overexpressed at the RPE and astrocytes, but mostly disappeared from the OPL.