V. Torbidoni

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 (&Dgr;ROS 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 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.

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.

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.

Endothelin Receptors : Do They Have a Role in Retinal Degeneration?

Although endothelins (ETs) were originally discovered as potent vasoconstrictors secreted by the endothelium (Yanagisawa et al., 1988), it has become evident that they also fulfill important roles in the nervous system (Schinelli, 2006). ETs are a family of 21-amino-acid peptides with three isoforms, ET-1, ET-2 and ET-3. ET-1 and ET-3 are present in most ocular structures. They are present in most regions of the anterior segment and in blood vessels of the choroid and the sclera (Chakrabarti and Sima, 1997). Precursor mRNAs of the three endothelins have been detected in the retina (Chakrabarti et al., 1998; Rattner and Nathans, 2005). ET-1 and ET-3 precursor mRNA appear in several retinal cell phenotypes (Ripodas et al., 2001), but the largest protein concentration appears in astrocytes (Torbidoni et al., 2005). ETs mediate their action through binding to two G-protein-linked receptors, ET-A and ET-B, which have been isolated and cloned from mammalian tissues (Davenport and Maguire, 2006). ET-A is characterized by the rank affinity order ET-1=ET-2>ET-3 whereas ET-B accepts all ET isopeptides with similar affinity (Kis et al., 2006). Activation of ET receptors triggers various shortand long-term changes at cellular level but their effects on brain and retinal physiology and pathophysiology are still poorly understood, since the ET system can behave completely differently in each cell type. Complex interactions occur between both endothelinergic receptors at different levels of the signaling pathways (Blomstrand et al., 2004), and it can sometimes be difficult to recognize receptor-specific responses. ET-B, however, is unique in its ability to internalize bound ligand, reducing extracellular levels of ETs (D’Orleans-Juste et al., 2002). Brain astrocytes are major endothelinergic effectors. ET-1 has been implicated in astrocytic regulation of the blood-brain barrier (Abbott et al., 2006), gap junctional coupling, proliferation, and pathological responses (Ostrow and