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Control of the retinal local RAS by the RPE: An interface to systemic RAS activity
Experimental Eye Research 189 (2019) 107838
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Control of the retinal local RAS by the RPE: An interface to systemic RAS activity
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Nadine Reichhart, Aleksandar Figura, Sergej Skosyrski, Olaf Strauß∗ Experimental Ophthalmology, Department of Ophthalmology, Charité – Universitätsmedizin Berlin, A Corporate Member of Freie Universität, Humboldt-University, The Berlin Institute of Health, Berlin, Germany
A B S T R A C T
As many other organs, the retina has a local renin-angiotensin-system (RAS). All main elements of the RAS are active in the retina: renin, angiotensinogen, angiotensin-converting enzymes. The functional role of the intraretinal RAS is not fully understood. So far, histological and functional analysis point to a regulation of ganglion cell activity and maybe also of bipolar cell activity, but it is not clear how RAS contributes to retinal signal processing. In contrast to local RAS in other organs, the retinal RAS is clearly separated from the systemic RAS. The angiotensin-2 (AngII)/AngI ratio in the retina is different to that in the plasma. However, it appears that the retinal pigment epithelium (RPE), that forms the outer blood/retina barrier, is a major regulator of the retinal RAS by producing renin. Interestingly, comparable to the kidney, the renin production in the RPE is under control of the angiotensin-2 receptor type-1 (AT1). AT1 localizes to the basolateral membrane of the RPE and faces the blood side of the blood/retina barrier. Increases in systemic AngII reduce renin production in the RPE and therefore decrease the intraretinal RAS activity. The relevance of the local RAS for retinal function remains unclear. Nevertheless, it is of fundamental significance to understand the pathology of systemically induced retinal diseases such as hypertension or diabetes.
1. Introduction The retinal pigment epithelium (RPE) is a close interaction partner of photoreceptors and represents a tight epithelium that forms the outer blood retina barrier (Bok, 1993; Miller and Steinberg, 1977; Steinberg, 1985; Strauss, 2005). The interaction of the RPE with the photoreceptors and its contribution to visual function is well investigated (Strauss, 2005). However, RPEs’ role as the active outer retina blood/ retina barrier, representing an interface with the body system, is less investigated. This knowledge, however, is important to understand the etiology of retinal diseases as the RPE faces changes in the body system, among them pathologic ones such as hypertension or diabetes. Several lines of evidence show that the RPE is equipped with receptors that react to factors that are present in the blood stream. For example, the RPE functions as part of the immune system in a way that it forms an active immune-inhibitory barrier by secretion of immune inhibitory factors (Streilein, 2003; Streilein et al., 2002). By this immune inhibition, the RPE establishes the immune privilege of the retina. The immune inhibitory phenotype can change in disease towards a pro-inflammatory one that promotes disease pathology or neovascularization. Adrenergic receptors adapt the transepithelial transport of water driven by a Cl− transport in the same direction across the RPE (Edelman and Miller, 1991; Joseph and Miller, 1992; Quinn et al., 2001; Rymer et al., 2001). Work from Jurklies, Jacobi and colleagues ((Jacobi et al., 1994b;
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Jurklies et al., 1995; Kohler et al., 1997) showed intriguingly that inhibition of the systemic RAS by systemic application of blockers for angiotensin-converting enzyme (ACE) reduces signals of the Ganzfeld electroretinogram (ERG), indicating that the systemic RAS influences neuronal activity of the retina. Work by our group shows that the systemic RAS activity influences the local retinal RAS activity via renin production in the RPE (Milenkovic et al., 2010). In the following, this article discusses the local retinal RAS and the implications of its systemic regulation by systemic impacts. 1.1. RAS in the retina In 1989, the first paper was published that indicated the existence of a local RAS in the retina. Deinum and colleagues reported local expression of renin in the eye and in the retina (Deinum et al., 1989, 1990). In the next years, other components of the RAS were reported and finally the groundbreaking publication by Wagner et al. (1996) demonstrated that all components of the RAS could be detected in the retina. Later, other groups that showed protein expression or even protease activities of ACE or renin, verified these data (Kohler et al., 1997; Luhtala et al., 2009; Milenkovic et al., 2010; Murata et al., 1997; Senanayake et al., 2007; Tikellis et al., 2004; Wheeler-Schilling et al., 2001). Furthermore, Danser et al. (1994) measured AngI and AngII levels in the eye and in the plasma and found that the retinal RAS
Corresponding author. E-mail address: [email protected] (O. Strauß).
https://doi.org/10.1016/j.exer.2019.107838 Received 7 May 2019; Received in revised form 19 August 2019; Accepted 11 October 2019 Available online 14 October 2019 0014-4835/ © 2019 Elsevier Ltd. All rights reserved.
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activity differs from that of the plasma indicating that the retinal RAS acts independently from systemic RAS and might execute retina-specific functions. Thus, the retina adds to the number of tissues that express local RAS. In contrast to other RAS expressing organs, the retinal RAS is separated from the systemic RAS by the blood/retina barrier which is formed by the RPE in the outer retina and endothelial cells of blood vessels in the inner retina (Fletcher et al., 2010). The largest body of evidence about the relevance of the retinal RAS demonstrated its role in diabetic retinopathy or hypertensive retinopathy (Reichhart et al., 2016; Wilkinson-Berka, 2004, 2006; Wilkinson-Berka et al., 2012). Here, modulation of the retinal RAS is a promising target to address retinal degeneration in these diseases. 1.2. The renin production in the retina: RPE cells As mentioned above, the retina contains quite high concentrations of renin. In fact, the renin protease activity measured in the mouse retina is comparable to that of the mouse plasma (Milenkovic et al., 2010). Renin is the protease that converts angiotensinogen into AngI and therefore ignites the reaction cascade of the RAS. Thus, cells that produce renin are the regulators of the retinal RAS. mRNA detection revealed that both Müller cells and RPE cells express renin (Berka et al., 1995; Brandt et al., 1994; Deinum et al., 1989; Milenkovic et al., 2010). Renin protein was found so far in the RPE (Milenkovic et al., 2010) and prorenin/renin in Müller cells (Berka et al., 1995; Fletcher et al., 2010). Thus, Müller cells and the RPE are the main regulators of the retinal RAS. Since retinal RAS represents a target to treat retinal degeneration, it is crucial for therapy development to understand how renin production is regulated and how patho-mechanisms increase renin production (Wilkinson-Berka et al., 2010). The ability of the RPE to produce renin enables a completely new understanding of the barrier function of the RPE. In the retina, the RAS activity, measured as the AngII/AngI ratio, appeared to be independent to that of the RAS in the plasma (Danser et al., 1994). Analysis of the renin gene expression in mouse under different systemic conditions showed astonishing results. The renin production in the RPE decreased after 24 h of water deprivation whereas that in the kidney increased (Milenkovic et al., 2010). In contrast, systemic application of the ACE inhibitor enalapril increased the renin expression in the kidney as well as in the retina. A separated analysis of the renin expression between retina and RPE showed that the retina increased the renin production even stronger than the RPE in response to the ACE inhibitor. These data imply that AngII in the plasma might be the strong regulator of the renin expression of the RPE and might act in a comparable manner to the kidney. Indeed, AngII infusion via micropumps in mice strongly reduced renin expression in the RPE (Milenkovic et al., 2010). When mice received the AT1 blocker losartan via drinking water during AngII infusion, the renin expression remained unchanged in the RPE. Thus, AngII in the plasma regulates the renin production by the RPE and therefore the intraretinal RAS via activation of AT1. This conclusion is supported by the observation that the RPE functionally expresses AT1 and that AT1 localizes to the basolateral membrane of the RPE facing the blood side of the outer blood/retina barrier. It is likely that an increase in intracellular free Ca2+, evoked by AngII binding to the AT1 receptors, is the second messenger that inhibits the renin expression in the RPE. A more detailed analysis showed that this Ca2+-signal depends on the activation of angiotensin-receptor-associated protein (ATRAP), release of Ca2+ from cytosolic Ca2+ stores and the activation of transient receptor potential vanilloid subtype-2 (TRPV2) Ca2+ channels in the RPE plasma membrane (Barro-Soria et al., 2012). Recently, we reported another regulatory mechanism for renin expression in the RPE that is based on adrenergic receptor activation (Strauss et al., 2013) Martins, 2019. Detection of adrenergic nerve endings in the choroid (Lutjen-Drecoll, 2006) that are tyrosine hydroxylase positive (Strauss et al., 2013) Martins, 2019 and thus adrenaline/noradrenaline production sites close to the RPE, led us to the hypothesis that the RPE
Fig. 1. The role of the RPE for the local retinal RAS: changes in systemic RAS are detected by basolateral AT1 receptors of the RPE. Once AngII has bound to AT1, an increase in intracellular free Ca2+ is generated via involvement of ATRAP and TRPV2 Ca2+ conducting channels. Increases in intracellular free Ca2+ reduce renin expression in the RPE resulting in a decrease of the retinal RAS activity. Retinal AngII levels decrease and neuronal activity in the retina, most likely of ganglion cells and bipolar cells is reduced. On the other hand, high retinal AngII levels contribute to neuronal cell death and vascular pathology. (AngII = angiotensin-2; ACE = angiotensin converting enzyme; ATRAP = angiotensin-receptor associated protein; RAS = renin angiotensin system; TRPV2 = transient receptor potential channel – vanilloid subtype-2).
might be under influence of systemic adrenergic stimulation. Isoproterenol infusion in mice led to a strong increase of renin expression in the RPE but not in the retina. Furthermore, the renin expression level in the RPE in presence of losartan was the same with or without isoproterenol. Thus, adrenergic increase of renin expression in the RPE does not occur via adrenergic inhibition of AT1-dependent reduction in renin expression. However, a cross talk between the signaling pathways might exist. In cultured porcine RPE, the effects of adrenergic stimulation could be mimicked by application of a cocktail of IBMX and forskolin to increase cytosolic cAMP as second messenger. Analysis of the renin promotor activity further revealed that the activity was increased by IBMX/forskolin but remained unchanged under AngII stimulation. With these data, the regulation of the renin expression in the RPE is comparable to that in the kidney. In both tissues, the mechanism that decreases renin mRNA by AngII is not understood. Changes of the promotor activity elicited by AngII, however, can be excluded. The regulation of renin expression in the RPE by systemic RAS has intriguing consequences for the retinal local RAS (Fig. 1). As discussed above, the increased systemic RAS activity in response to loss of extracellular volume by water deprivation decreases retinal RAS activity. Considering a physiological role of the retinal RAS, this effect would result in a systemic regulation of specific functions in the retina (Fletcher et al., 2010). The type of retinal function that is altered by systemically inhibited renin production in the RPE is not clear; it might be either homeostatic control of retinal function or direct modulation of retinal signal processing (Fletcher et al., 2010). The concept that systemic RAS regulates retinal activity fits into the hypothesis that increased systemic levels of AngII reduce renin production in the RPE that in turn decreases retinal RAS and was proven by the experiments by Jurklies, Jacobi and colleagues (Jacobi et al., 1994a, 1994b; Jurklies et al., 1995). In these experiments, ACE inhibitors were systemically applied and retinal activity was measured by ERG. Systemic ACE inhibition changed amplitudes of the b-wave and oscillatory potentials of the Ganzfeld ERG. Small doses resulted in an acute increase in the bwave that returned to levels below baseline after an hour and did not 2
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of AngII on vascular function has been shown at different levels. Data indicate vasoconstriction by intracellular Ca2+ increases, as excellently reviewed by Fletcher et al. (2010). However, it is likely that in vivo, vascular reactions are predominantly evoked by changes of the systemic RAS and not of the local intraretinal RAS. In contrast, as the RPE and maybe Müller cells are able to produce renin, these cells predominantly control intraretinal RAS. Functional data might be obtained either by measurement of the electroretinogram (ERG) or by direct evaluation of AngII signaling in cells. The latter can be investigated by measurements of intracellular free Ca2+ or neuronal activity by means of the patchclamp technique. Jurklies/Jacobi's data showed changes in the b-wave and oscillatory potentials after systemic application of ACE inhibitors (Jacobi et al., 1994a, 1994b, 1996; Jurklies et al., 1995). As summarized above, systemic application of ACE inhibitors would result in an increase of the local retinal RAS activity in eyes with intact blood/retina barrier. The b-wave is a signal in the Ganzfeld-ERG that results from both the activity of ON bipolar cells and by the K+ buffering activity of Müller cells (Pardue and Peachey, 2014; Young et al., 2012). Oscillatory potentials are oscillations of the membrane voltage along the bipolar cells axis, induced by inhibitory feedback input from amacrine cells (Wachtmeister, 1998). Thus, these data support the data from immunohistochemical analysis that identified both cell types as potentially positive for AT1 and/or AT2. Given the unreliability of immunohistochemical AT1 staining, the additional information from ERG analysis cannot decipher the AngII receptor subtype on either Müller, amacrine or bipolar cells. Furthermore, subtypes of bipolar cells or amacrine cells that react directly to AngII stimulation were not yet identified. Concerning the role of AT1, so far only the analysis of the ganglion cells has revealed reliable data. As mentioned above, immunostaining with antibodies against AT1 that were tested in knockout tissue showed that ganglion cells are positive for AT1. Patch-clamp analysis of freshly isolated ganglion cells showed modulation of voltage-dependent Ca2+ channels upon stimulation by AngII (Guenther et al., 1996, 1997). Thus, it is likely that AngII changes ganglion cell activity via AT1 stimulation. The nature of this effect depends on the subtype of ganglion cell, especially on the expression levels of L-type and N-type voltage-gated Ca2+ channels. Whereas AngII generally reduces the activity of the synaptic N-type channel, it might either activate or inhibit L-type channels at the cell soma. Whether ON or OFF ganglion cells account for these differential effects is not clear. Unpublished data by our group support this conclusion by analysis of the photopic mouse ERG under influence of intra-ocularly applied AngII or the AT1 blocker losartan (Fig. 2). In the case of intraocular injection of an AngII containing solution, acute activation of retinal RAS is mimicked. As discussed above, this condition would also correspond to the systemic ACE inhibition in experiments by Jurklies/Jacobi. Application of AngII influences neither the a-wave (corresponds with cone activity) nor the b-wave of the photopic Ganzfeld-ERG. Specific changes, however, were observed in the pattern ERG (pERG). pERG is a measure of retinal electric activity that is stimulated by patterns of various sizes. This method permits monitoring the activity of ganglion cells. The main effect of intraocular AngII injection was an increase in the latency of the P50 without affecting the amplitude. This result might point towards a reduced ganglion cell activity (Harrison et al., 1987), although an influence of neuronal activity upstream to ganglion cells cannot be excluded. Thus, AngII injection experiments show that the increase in RAS mostly affects the signaling of ganglion cells. More puzzling are the data from intraocular injections of the AT1 blocker losartan. This experiment analyzes AT1 blockade in the condition of resting RAS. Intraocular losartan application increased the b-wave in the photopic Ganzfeld-ERG without affecting cone activity, representing a functional role of AT1 or AT2 in bipolar cells and Müller cells. Again, these data cannot clarify the differential functional relevance of the receptor subtypes. It might be that the competitive blocker for AT1 increases the activity of AT2 because the majority of AngII binds now to AT2. Losartan affected also the pERG. Here, losartan reduced the P50 amplitude for the pattern
further change. Higher doses acutely reduced the b-wave amplitudes. In experiments that explored the effects of systemic ACE inhibition on RPE renin expression, mice were treated with a high dosage of ACE inhibitor for six days prior to measurement of renin mRNA. Thus, these experiments corresponded with the situation of a high dosage over a longer time (Milenkovic et al., 2010). Under these conditions, systemic ACE inhibition led to increased renin expression in the RPE and in consequence to a higher retinal RAS activity. Since these experiments correspond with that of high dosage and long exposure in the Jurklies/ Jacobi experiments (Jacobi et al., 1994a, 1994b; Jurklies et al., 1995), it is likely that increased systemic RAS activity leads to lower retinal RAS activity which in turn increases neuronal activity in the retina. Another effect of the control of retinal RAS by the RPE can be seen in the pathology of systemically induced retinal degenerations (Reichhart et al., 2016; Wilkinson-Berka, 2004, 2006; Wilkinson-Berka et al., 2012; Williams et al., 2013; Wong and Mitchell, 2007). For example, high retinal RAS activity contributes to retinal degeneration in hypertensive or diabetic retinopathy (Fukuda et al., 2010; Narimatsu et al., 2014; Reichhart et al., 2016; White et al., 2015). In case the RPE barrier function is intact, a pharmacologically induced reduction in systemic AngII by for example ACE inhibitors would increase the local RAS in the retina and foster AngII-dependent patho-mechanisms in retinal degeneration. Thus, treatment of diabetic patients with ACE inhibitors would increase AngII-dependent damage. On the other hand, systemic application of AT1 blockers would uncouple RPE's renin production from systemic RAS. These effects, however, warrant further evaluation in clinical settings. 1.3. The functional role of retinal RAS The relevance of the regulation of renin expression in the RPE is determined by the functional role of the RAS in the retina. As discussed above, this might be either the regulation of neuronal activity or the control of tissue homeostasis such as volume control or both. The RAS has two main reaction end-products that mediate RAS functions: AngII (Ang1-8) activates AT1 and AT2 whereas Ang1-7 activates MAS receptors. Thus, identification of retinal cells that carry one of these receptors might provide hints for the functional role of the RAS in the retina. Histological analysis to detect the target cells of either AngII or Ang1-7 is not easy and produced contradictory results (Danser et al., 1994; Datum and Zrenner, 1991; Senanayake et al., 2007). One option is the detection of cells that are positive for AngII. Intracellular AngII might result from internalization of AT1 together with AngII once AngII has bound to AT1. Using this technique, Müller cells (Senanayake et al., 2007) and amacrine cells appeared positive for AngII (Datum and Zrenner, 1991; Kohler et al., 1997). Since Müller cells, also showed the cleavage product Ang1-7, it is possible that Müller cells are rather regulators of the retinal RAS (Senanayake et al., 2007). Radiolabeling of AngII showed that retinal endothelial cells express AT1 receptors, too (Downie et al., 2009; Ferrari-Dileo et al., 1987). Using antibodies against AT1 is known to lead to rather non-reliable results. Due to AT1's ability to change its extracellular three-dimensional structure, generating different binding patterns for AngII (Balakumar and Jagadeesh, 2014), antibodies developed against AT1 are rarely specific. Using these antibodies a couple of cells were identified that might express AT1: astrocytes and Müller cells and a certain subset of bipolar cells (Datum and Zrenner, 1991; Downie et al., 2009; Fletcher et al., 2010; Milenkovic et al., 2010; Senanayake et al., 2007). However, using an antibody validated in the kidney of AT1/2 double knock-out mice, the paper by Milenkovic et al. showed AT1 staining only in ganglion cells and in the RPE (Milenkovic et al., 2010). Since the group analyzed only sagittal sections of the retina, it might be that the positive staining of blood vessels was missed. AT2 staining was found in different retinal neurons, among them amacrine cells and a subgroup of bipolar cells (Downie et al., 2009). These data can only be verified by functional analysis. The influence 3
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Fig. 2. Reaction of retinal activity to intraocular AngII (100 nM) or losartan (5 μM) application assessed by ERG techniques: A: photopic Ganzfeld-ERG: mean values of single flash responses from 7 eyes at the intensity of 20cds/m2; red = AngII injection; black = 0.9% NaCl control. B: Statistical comparison of b-wave amplitudes of AngII (white) and NaCl control (black) for the flash intensities 5 and 20 cds/m2. C: patternERG: mean values from 15 eyes at a pattern size of 15°; red = AngII; black = PBS control. D: patternERG: mean values from 8 eyes at a pattern size of 10°; red = AngII; black = PBS control. E: statistical analysis of the latency to reach the P50 at the patternERG for several pattern sizes; AngII = white and PBS control = black. F: photopic Ganzfeld-ERG: mean values of single flash responses from 5 eyes at the intensity of 20cds/m2; red = losartan injection; black = NaCl control. G: Statistical comparison of b-wave amplitude of losartan (white) and NaCl control (black) for the flash intensities 5 and 20 cds/m2. H: patternERG: mean values from 15 eyes at a pattern size of 15°; red = losartan; black = NaCl control. I: patternERG: mean values from 10 eyes at a pattern size of 10°; red = AngII; black = NaCl control. E: statistical analysis of the latency to reach the P50 at the patternERG for several pattern sizes; AngII = white and PBS control = black. All data are given as mean ± SEM. *p < 0.05; ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 4
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sizes between 15° and 6°. Since the bipolar cell activity might be affected by losartan and since it is likely that losartan also affects ganglion cell activity, the losartan-dependent changes in the pERG result from a mixture of both sites of action. Indeed, we found significant increased P50 amplitudes at pattern sizes between 15° and 8°. In summary, it is likely that the local retinal RAS is a regulator of neuronal activity in the retina.
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1.4. A hypothesis for the regulation of the retinal RAS by the RPE The above-described data strongly suggest that the retinal RAS regulates neuronal activity by either AT1 and/or AT2 stimulation. AngII-dependent activation of AT1 appeared to reduce neuronal activity as observed in ganglion cells. The main regulators of the retinal RAS are the renin producing Müller cells and RPE cells. The regulation of retinal RAS by the RPE is under strong influence by systemic RAS or by systemic adrenergic activity. Thus, the RPE-dependent regulation of retinal RAS represents a mechanism, by which retinal signal processing can be adapted to changes in the body system. For example, water deprivation increases RPE's renin production and influences the activity of ganglion cells. The physiological relevance of this effect is completely unknown. The patho-physiological relevance is highlighted by the large number of studies that indicate the role of retinal RAS in systemically induced retinal degenerations. Acknowledgements The work was supported by a research grant of the Ernst and Berta Grimmke Stiftung, Düsseldorf Germany. References Balakumar, P., Jagadeesh, G., 2014. Structural determinants for binding, activation, and functional selectivity of the angiotensin AT1 receptor. J. Mol. Endocrinol. 53, R71–R92. Barro-Soria, R., Stindl, J., Muller, C., Foeckler, R., Todorov, V., Castrop, H., Strauss, O., 2012. Angiotensin-2-mediated Ca2+ signaling in the retinal pigment epithelium: role of angiotensin-receptor-associated-protein and TRPV2 channel. PLoS One 7, e49624. Berka, J.L., Stubbs, A.J., Wang, D.Z., DiNicolantonio, R., Alcorn, D., Campbell, D.J., Skinner, S.L., 1995. Renin-containing Muller cells of the retina display endocrine features. Investig. Ophthalmol. Vis. Sci. 36, 1450–1458. Bok, D., 1993. The retinal pigment epithelium: a versatile partner in vision. J. Cell Sci. Suppl. 17, 189–195. Brandt, C.R., Pumfery, A.M., Micales, B., Bindley, C.D., Lyons, G.E., Sramek, S.J., Wallow, I.H., 1994. Renin mRNA is synthesized locally in rat ocular tissues. Curr. Eye Res. 13, 755–763. Danser, A.H., Derkx, F.H., Admiraal, P.J., Deinum, J., de Jong, P.T., Schalekamp, M.A., 1994. Angiotensin levels in the eye. Investig. Ophthalmol. Vis. Sci. 35, 1008–1018. Datum, K.H., Zrenner, E., 1991. Angiotensin-like immunoreactive cells in the chicken retina. Exp. Eye Res. 53, 157–165. Deinum, J., Derkx, F.H., Danser, A.H., Schalekamp, M.A., 1989. Renin in the bovine eye. J. Hypertens. Suppl. 7, S216–S217. Deinum, J., Derkx, F.H., Danser, A.H., Schalekamp, M.A., 1990. Identification and quantification of renin and prorenin in the bovine eye. Endocrinology 126, 1673–1682. Downie, L.E., Vessey, K., Miller, A., Ward, M.M., Pianta, M.J., Vingrys, A.J., WilkinsonBerka, J.L., Fletcher, E.L., 2009. Neuronal and glial cell expression of angiotensin II type 1 (AT1) and type 2 (AT2) receptors in the rat retina. Neuroscience 161, 195–213. Edelman, J.L., Miller, S.S., 1991. Epinephrine stimulates fluid absorption across bovine retinal pigment epithelium. Investig. Ophthalmol. Vis. Sci. 32, 3033–3040. Ferrari-Dileo, G., Davis, E.B., Anderson, D.R., 1987. Angiotensin binding sites in bovine and human retinal blood vessels. Investig. Ophthalmol. Vis. Sci. 28, 1747–1751. Fletcher, E.L., Phipps, J.A., Ward, M.M., Vessey, K.A., Wilkinson-Berka, J.L., 2010. The renin-angiotensin system in retinal health and disease: its influence on neurons, glia and the vasculature. Prog. Retin. Eye Res. 29, 284–311. Fukuda, K., Hirooka, K., Mizote, M., Nakamura, T., Itano, T., Shiraga, F., 2010. Neuroprotection against retinal ischemia-reperfusion injury by blocking the angiotensin II type 1 receptor. Investig. Ophthalmol. Vis. Sci. 51, 3629–3638. Guenther, E., Hewig, B., Zrenner, E., Kohler, K., 1997. Angiotensin II-induced inhibition and facilitation of calcium current subtypes in rat retinal ganglion cells. Neurosci. Lett. 231, 71–74. Guenther, E., Schmid, S., Hewig, B., Kohler, K., 1996. Two-fold effect of Angiotensin II on voltage-dependent calcium currents in rat retinal ganglion cells. Brain Res. 718, 112–116. Harrison, J.M., O'Connor, P.S., Young, R.S., Kincaid, M., Bentley, R., 1987. The pattern ERG in man following surgical resection of the optic nerve. Investig. Ophthalmol. Vis.
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Control of the retinal local RAS by the RPE: An interface to systemic RAS activity
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Nadine Reichhart, Aleksandar Figura, Sergej Skosyrski, Olaf Strauß∗ Experimental Ophthalmology, Department of Ophthalmology, Charité – Universitätsmedizin Berlin, A Corporate Member of Freie Universität, Humboldt-University, The Berlin Institute of Health, Berlin, Germany
A B S T R A C T
As many other organs, the retina has a local renin-angiotensin-system (RAS). All main elements of the RAS are active in the retina: renin, angiotensinogen, angiotensin-converting enzymes. The functional role of the intraretinal RAS is not fully understood. So far, histological and functional analysis point to a regulation of ganglion cell activity and maybe also of bipolar cell activity, but it is not clear how RAS contributes to retinal signal processing. In contrast to local RAS in other organs, the retinal RAS is clearly separated from the systemic RAS. The angiotensin-2 (AngII)/AngI ratio in the retina is different to that in the plasma. However, it appears that the retinal pigment epithelium (RPE), that forms the outer blood/retina barrier, is a major regulator of the retinal RAS by producing renin. Interestingly, comparable to the kidney, the renin production in the RPE is under control of the angiotensin-2 receptor type-1 (AT1). AT1 localizes to the basolateral membrane of the RPE and faces the blood side of the blood/retina barrier. Increases in systemic AngII reduce renin production in the RPE and therefore decrease the intraretinal RAS activity. The relevance of the local RAS for retinal function remains unclear. Nevertheless, it is of fundamental significance to understand the pathology of systemically induced retinal diseases such as hypertension or diabetes.
1. Introduction The retinal pigment epithelium (RPE) is a close interaction partner of photoreceptors and represents a tight epithelium that forms the outer blood retina barrier (Bok, 1993; Miller and Steinberg, 1977; Steinberg, 1985; Strauss, 2005). The interaction of the RPE with the photoreceptors and its contribution to visual function is well investigated (Strauss, 2005). However, RPEs’ role as the active outer retina blood/ retina barrier, representing an interface with the body system, is less investigated. This knowledge, however, is important to understand the etiology of retinal diseases as the RPE faces changes in the body system, among them pathologic ones such as hypertension or diabetes. Several lines of evidence show that the RPE is equipped with receptors that react to factors that are present in the blood stream. For example, the RPE functions as part of the immune system in a way that it forms an active immune-inhibitory barrier by secretion of immune inhibitory factors (Streilein, 2003; Streilein et al., 2002). By this immune inhibition, the RPE establishes the immune privilege of the retina. The immune inhibitory phenotype can change in disease towards a pro-inflammatory one that promotes disease pathology or neovascularization. Adrenergic receptors adapt the transepithelial transport of water driven by a Cl− transport in the same direction across the RPE (Edelman and Miller, 1991; Joseph and Miller, 1992; Quinn et al., 2001; Rymer et al., 2001). Work from Jurklies, Jacobi and colleagues ((Jacobi et al., 1994b;
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Jurklies et al., 1995; Kohler et al., 1997) showed intriguingly that inhibition of the systemic RAS by systemic application of blockers for angiotensin-converting enzyme (ACE) reduces signals of the Ganzfeld electroretinogram (ERG), indicating that the systemic RAS influences neuronal activity of the retina. Work by our group shows that the systemic RAS activity influences the local retinal RAS activity via renin production in the RPE (Milenkovic et al., 2010). In the following, this article discusses the local retinal RAS and the implications of its systemic regulation by systemic impacts. 1.1. RAS in the retina In 1989, the first paper was published that indicated the existence of a local RAS in the retina. Deinum and colleagues reported local expression of renin in the eye and in the retina (Deinum et al., 1989, 1990). In the next years, other components of the RAS were reported and finally the groundbreaking publication by Wagner et al. (1996) demonstrated that all components of the RAS could be detected in the retina. Later, other groups that showed protein expression or even protease activities of ACE or renin, verified these data (Kohler et al., 1997; Luhtala et al., 2009; Milenkovic et al., 2010; Murata et al., 1997; Senanayake et al., 2007; Tikellis et al., 2004; Wheeler-Schilling et al., 2001). Furthermore, Danser et al. (1994) measured AngI and AngII levels in the eye and in the plasma and found that the retinal RAS
Corresponding author. E-mail address: [email protected] (O. Strauß).
https://doi.org/10.1016/j.exer.2019.107838 Received 7 May 2019; Received in revised form 19 August 2019; Accepted 11 October 2019 Available online 14 October 2019 0014-4835/ © 2019 Elsevier Ltd. All rights reserved.
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activity differs from that of the plasma indicating that the retinal RAS acts independently from systemic RAS and might execute retina-specific functions. Thus, the retina adds to the number of tissues that express local RAS. In contrast to other RAS expressing organs, the retinal RAS is separated from the systemic RAS by the blood/retina barrier which is formed by the RPE in the outer retina and endothelial cells of blood vessels in the inner retina (Fletcher et al., 2010). The largest body of evidence about the relevance of the retinal RAS demonstrated its role in diabetic retinopathy or hypertensive retinopathy (Reichhart et al., 2016; Wilkinson-Berka, 2004, 2006; Wilkinson-Berka et al., 2012). Here, modulation of the retinal RAS is a promising target to address retinal degeneration in these diseases. 1.2. The renin production in the retina: RPE cells As mentioned above, the retina contains quite high concentrations of renin. In fact, the renin protease activity measured in the mouse retina is comparable to that of the mouse plasma (Milenkovic et al., 2010). Renin is the protease that converts angiotensinogen into AngI and therefore ignites the reaction cascade of the RAS. Thus, cells that produce renin are the regulators of the retinal RAS. mRNA detection revealed that both Müller cells and RPE cells express renin (Berka et al., 1995; Brandt et al., 1994; Deinum et al., 1989; Milenkovic et al., 2010). Renin protein was found so far in the RPE (Milenkovic et al., 2010) and prorenin/renin in Müller cells (Berka et al., 1995; Fletcher et al., 2010). Thus, Müller cells and the RPE are the main regulators of the retinal RAS. Since retinal RAS represents a target to treat retinal degeneration, it is crucial for therapy development to understand how renin production is regulated and how patho-mechanisms increase renin production (Wilkinson-Berka et al., 2010). The ability of the RPE to produce renin enables a completely new understanding of the barrier function of the RPE. In the retina, the RAS activity, measured as the AngII/AngI ratio, appeared to be independent to that of the RAS in the plasma (Danser et al., 1994). Analysis of the renin gene expression in mouse under different systemic conditions showed astonishing results. The renin production in the RPE decreased after 24 h of water deprivation whereas that in the kidney increased (Milenkovic et al., 2010). In contrast, systemic application of the ACE inhibitor enalapril increased the renin expression in the kidney as well as in the retina. A separated analysis of the renin expression between retina and RPE showed that the retina increased the renin production even stronger than the RPE in response to the ACE inhibitor. These data imply that AngII in the plasma might be the strong regulator of the renin expression of the RPE and might act in a comparable manner to the kidney. Indeed, AngII infusion via micropumps in mice strongly reduced renin expression in the RPE (Milenkovic et al., 2010). When mice received the AT1 blocker losartan via drinking water during AngII infusion, the renin expression remained unchanged in the RPE. Thus, AngII in the plasma regulates the renin production by the RPE and therefore the intraretinal RAS via activation of AT1. This conclusion is supported by the observation that the RPE functionally expresses AT1 and that AT1 localizes to the basolateral membrane of the RPE facing the blood side of the outer blood/retina barrier. It is likely that an increase in intracellular free Ca2+, evoked by AngII binding to the AT1 receptors, is the second messenger that inhibits the renin expression in the RPE. A more detailed analysis showed that this Ca2+-signal depends on the activation of angiotensin-receptor-associated protein (ATRAP), release of Ca2+ from cytosolic Ca2+ stores and the activation of transient receptor potential vanilloid subtype-2 (TRPV2) Ca2+ channels in the RPE plasma membrane (Barro-Soria et al., 2012). Recently, we reported another regulatory mechanism for renin expression in the RPE that is based on adrenergic receptor activation (Strauss et al., 2013) Martins, 2019. Detection of adrenergic nerve endings in the choroid (Lutjen-Drecoll, 2006) that are tyrosine hydroxylase positive (Strauss et al., 2013) Martins, 2019 and thus adrenaline/noradrenaline production sites close to the RPE, led us to the hypothesis that the RPE
Fig. 1. The role of the RPE for the local retinal RAS: changes in systemic RAS are detected by basolateral AT1 receptors of the RPE. Once AngII has bound to AT1, an increase in intracellular free Ca2+ is generated via involvement of ATRAP and TRPV2 Ca2+ conducting channels. Increases in intracellular free Ca2+ reduce renin expression in the RPE resulting in a decrease of the retinal RAS activity. Retinal AngII levels decrease and neuronal activity in the retina, most likely of ganglion cells and bipolar cells is reduced. On the other hand, high retinal AngII levels contribute to neuronal cell death and vascular pathology. (AngII = angiotensin-2; ACE = angiotensin converting enzyme; ATRAP = angiotensin-receptor associated protein; RAS = renin angiotensin system; TRPV2 = transient receptor potential channel – vanilloid subtype-2).
might be under influence of systemic adrenergic stimulation. Isoproterenol infusion in mice led to a strong increase of renin expression in the RPE but not in the retina. Furthermore, the renin expression level in the RPE in presence of losartan was the same with or without isoproterenol. Thus, adrenergic increase of renin expression in the RPE does not occur via adrenergic inhibition of AT1-dependent reduction in renin expression. However, a cross talk between the signaling pathways might exist. In cultured porcine RPE, the effects of adrenergic stimulation could be mimicked by application of a cocktail of IBMX and forskolin to increase cytosolic cAMP as second messenger. Analysis of the renin promotor activity further revealed that the activity was increased by IBMX/forskolin but remained unchanged under AngII stimulation. With these data, the regulation of the renin expression in the RPE is comparable to that in the kidney. In both tissues, the mechanism that decreases renin mRNA by AngII is not understood. Changes of the promotor activity elicited by AngII, however, can be excluded. The regulation of renin expression in the RPE by systemic RAS has intriguing consequences for the retinal local RAS (Fig. 1). As discussed above, the increased systemic RAS activity in response to loss of extracellular volume by water deprivation decreases retinal RAS activity. Considering a physiological role of the retinal RAS, this effect would result in a systemic regulation of specific functions in the retina (Fletcher et al., 2010). The type of retinal function that is altered by systemically inhibited renin production in the RPE is not clear; it might be either homeostatic control of retinal function or direct modulation of retinal signal processing (Fletcher et al., 2010). The concept that systemic RAS regulates retinal activity fits into the hypothesis that increased systemic levels of AngII reduce renin production in the RPE that in turn decreases retinal RAS and was proven by the experiments by Jurklies, Jacobi and colleagues (Jacobi et al., 1994a, 1994b; Jurklies et al., 1995). In these experiments, ACE inhibitors were systemically applied and retinal activity was measured by ERG. Systemic ACE inhibition changed amplitudes of the b-wave and oscillatory potentials of the Ganzfeld ERG. Small doses resulted in an acute increase in the bwave that returned to levels below baseline after an hour and did not 2
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of AngII on vascular function has been shown at different levels. Data indicate vasoconstriction by intracellular Ca2+ increases, as excellently reviewed by Fletcher et al. (2010). However, it is likely that in vivo, vascular reactions are predominantly evoked by changes of the systemic RAS and not of the local intraretinal RAS. In contrast, as the RPE and maybe Müller cells are able to produce renin, these cells predominantly control intraretinal RAS. Functional data might be obtained either by measurement of the electroretinogram (ERG) or by direct evaluation of AngII signaling in cells. The latter can be investigated by measurements of intracellular free Ca2+ or neuronal activity by means of the patchclamp technique. Jurklies/Jacobi's data showed changes in the b-wave and oscillatory potentials after systemic application of ACE inhibitors (Jacobi et al., 1994a, 1994b, 1996; Jurklies et al., 1995). As summarized above, systemic application of ACE inhibitors would result in an increase of the local retinal RAS activity in eyes with intact blood/retina barrier. The b-wave is a signal in the Ganzfeld-ERG that results from both the activity of ON bipolar cells and by the K+ buffering activity of Müller cells (Pardue and Peachey, 2014; Young et al., 2012). Oscillatory potentials are oscillations of the membrane voltage along the bipolar cells axis, induced by inhibitory feedback input from amacrine cells (Wachtmeister, 1998). Thus, these data support the data from immunohistochemical analysis that identified both cell types as potentially positive for AT1 and/or AT2. Given the unreliability of immunohistochemical AT1 staining, the additional information from ERG analysis cannot decipher the AngII receptor subtype on either Müller, amacrine or bipolar cells. Furthermore, subtypes of bipolar cells or amacrine cells that react directly to AngII stimulation were not yet identified. Concerning the role of AT1, so far only the analysis of the ganglion cells has revealed reliable data. As mentioned above, immunostaining with antibodies against AT1 that were tested in knockout tissue showed that ganglion cells are positive for AT1. Patch-clamp analysis of freshly isolated ganglion cells showed modulation of voltage-dependent Ca2+ channels upon stimulation by AngII (Guenther et al., 1996, 1997). Thus, it is likely that AngII changes ganglion cell activity via AT1 stimulation. The nature of this effect depends on the subtype of ganglion cell, especially on the expression levels of L-type and N-type voltage-gated Ca2+ channels. Whereas AngII generally reduces the activity of the synaptic N-type channel, it might either activate or inhibit L-type channels at the cell soma. Whether ON or OFF ganglion cells account for these differential effects is not clear. Unpublished data by our group support this conclusion by analysis of the photopic mouse ERG under influence of intra-ocularly applied AngII or the AT1 blocker losartan (Fig. 2). In the case of intraocular injection of an AngII containing solution, acute activation of retinal RAS is mimicked. As discussed above, this condition would also correspond to the systemic ACE inhibition in experiments by Jurklies/Jacobi. Application of AngII influences neither the a-wave (corresponds with cone activity) nor the b-wave of the photopic Ganzfeld-ERG. Specific changes, however, were observed in the pattern ERG (pERG). pERG is a measure of retinal electric activity that is stimulated by patterns of various sizes. This method permits monitoring the activity of ganglion cells. The main effect of intraocular AngII injection was an increase in the latency of the P50 without affecting the amplitude. This result might point towards a reduced ganglion cell activity (Harrison et al., 1987), although an influence of neuronal activity upstream to ganglion cells cannot be excluded. Thus, AngII injection experiments show that the increase in RAS mostly affects the signaling of ganglion cells. More puzzling are the data from intraocular injections of the AT1 blocker losartan. This experiment analyzes AT1 blockade in the condition of resting RAS. Intraocular losartan application increased the b-wave in the photopic Ganzfeld-ERG without affecting cone activity, representing a functional role of AT1 or AT2 in bipolar cells and Müller cells. Again, these data cannot clarify the differential functional relevance of the receptor subtypes. It might be that the competitive blocker for AT1 increases the activity of AT2 because the majority of AngII binds now to AT2. Losartan affected also the pERG. Here, losartan reduced the P50 amplitude for the pattern
further change. Higher doses acutely reduced the b-wave amplitudes. In experiments that explored the effects of systemic ACE inhibition on RPE renin expression, mice were treated with a high dosage of ACE inhibitor for six days prior to measurement of renin mRNA. Thus, these experiments corresponded with the situation of a high dosage over a longer time (Milenkovic et al., 2010). Under these conditions, systemic ACE inhibition led to increased renin expression in the RPE and in consequence to a higher retinal RAS activity. Since these experiments correspond with that of high dosage and long exposure in the Jurklies/ Jacobi experiments (Jacobi et al., 1994a, 1994b; Jurklies et al., 1995), it is likely that increased systemic RAS activity leads to lower retinal RAS activity which in turn increases neuronal activity in the retina. Another effect of the control of retinal RAS by the RPE can be seen in the pathology of systemically induced retinal degenerations (Reichhart et al., 2016; Wilkinson-Berka, 2004, 2006; Wilkinson-Berka et al., 2012; Williams et al., 2013; Wong and Mitchell, 2007). For example, high retinal RAS activity contributes to retinal degeneration in hypertensive or diabetic retinopathy (Fukuda et al., 2010; Narimatsu et al., 2014; Reichhart et al., 2016; White et al., 2015). In case the RPE barrier function is intact, a pharmacologically induced reduction in systemic AngII by for example ACE inhibitors would increase the local RAS in the retina and foster AngII-dependent patho-mechanisms in retinal degeneration. Thus, treatment of diabetic patients with ACE inhibitors would increase AngII-dependent damage. On the other hand, systemic application of AT1 blockers would uncouple RPE's renin production from systemic RAS. These effects, however, warrant further evaluation in clinical settings. 1.3. The functional role of retinal RAS The relevance of the regulation of renin expression in the RPE is determined by the functional role of the RAS in the retina. As discussed above, this might be either the regulation of neuronal activity or the control of tissue homeostasis such as volume control or both. The RAS has two main reaction end-products that mediate RAS functions: AngII (Ang1-8) activates AT1 and AT2 whereas Ang1-7 activates MAS receptors. Thus, identification of retinal cells that carry one of these receptors might provide hints for the functional role of the RAS in the retina. Histological analysis to detect the target cells of either AngII or Ang1-7 is not easy and produced contradictory results (Danser et al., 1994; Datum and Zrenner, 1991; Senanayake et al., 2007). One option is the detection of cells that are positive for AngII. Intracellular AngII might result from internalization of AT1 together with AngII once AngII has bound to AT1. Using this technique, Müller cells (Senanayake et al., 2007) and amacrine cells appeared positive for AngII (Datum and Zrenner, 1991; Kohler et al., 1997). Since Müller cells, also showed the cleavage product Ang1-7, it is possible that Müller cells are rather regulators of the retinal RAS (Senanayake et al., 2007). Radiolabeling of AngII showed that retinal endothelial cells express AT1 receptors, too (Downie et al., 2009; Ferrari-Dileo et al., 1987). Using antibodies against AT1 is known to lead to rather non-reliable results. Due to AT1's ability to change its extracellular three-dimensional structure, generating different binding patterns for AngII (Balakumar and Jagadeesh, 2014), antibodies developed against AT1 are rarely specific. Using these antibodies a couple of cells were identified that might express AT1: astrocytes and Müller cells and a certain subset of bipolar cells (Datum and Zrenner, 1991; Downie et al., 2009; Fletcher et al., 2010; Milenkovic et al., 2010; Senanayake et al., 2007). However, using an antibody validated in the kidney of AT1/2 double knock-out mice, the paper by Milenkovic et al. showed AT1 staining only in ganglion cells and in the RPE (Milenkovic et al., 2010). Since the group analyzed only sagittal sections of the retina, it might be that the positive staining of blood vessels was missed. AT2 staining was found in different retinal neurons, among them amacrine cells and a subgroup of bipolar cells (Downie et al., 2009). These data can only be verified by functional analysis. The influence 3
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Fig. 2. Reaction of retinal activity to intraocular AngII (100 nM) or losartan (5 μM) application assessed by ERG techniques: A: photopic Ganzfeld-ERG: mean values of single flash responses from 7 eyes at the intensity of 20cds/m2; red = AngII injection; black = 0.9% NaCl control. B: Statistical comparison of b-wave amplitudes of AngII (white) and NaCl control (black) for the flash intensities 5 and 20 cds/m2. C: patternERG: mean values from 15 eyes at a pattern size of 15°; red = AngII; black = PBS control. D: patternERG: mean values from 8 eyes at a pattern size of 10°; red = AngII; black = PBS control. E: statistical analysis of the latency to reach the P50 at the patternERG for several pattern sizes; AngII = white and PBS control = black. F: photopic Ganzfeld-ERG: mean values of single flash responses from 5 eyes at the intensity of 20cds/m2; red = losartan injection; black = NaCl control. G: Statistical comparison of b-wave amplitude of losartan (white) and NaCl control (black) for the flash intensities 5 and 20 cds/m2. H: patternERG: mean values from 15 eyes at a pattern size of 15°; red = losartan; black = NaCl control. I: patternERG: mean values from 10 eyes at a pattern size of 10°; red = AngII; black = NaCl control. E: statistical analysis of the latency to reach the P50 at the patternERG for several pattern sizes; AngII = white and PBS control = black. All data are given as mean ± SEM. *p < 0.05; ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 4
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sizes between 15° and 6°. Since the bipolar cell activity might be affected by losartan and since it is likely that losartan also affects ganglion cell activity, the losartan-dependent changes in the pERG result from a mixture of both sites of action. Indeed, we found significant increased P50 amplitudes at pattern sizes between 15° and 8°. In summary, it is likely that the local retinal RAS is a regulator of neuronal activity in the retina.
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1.4. A hypothesis for the regulation of the retinal RAS by the RPE The above-described data strongly suggest that the retinal RAS regulates neuronal activity by either AT1 and/or AT2 stimulation. AngII-dependent activation of AT1 appeared to reduce neuronal activity as observed in ganglion cells. The main regulators of the retinal RAS are the renin producing Müller cells and RPE cells. The regulation of retinal RAS by the RPE is under strong influence by systemic RAS or by systemic adrenergic activity. Thus, the RPE-dependent regulation of retinal RAS represents a mechanism, by which retinal signal processing can be adapted to changes in the body system. For example, water deprivation increases RPE's renin production and influences the activity of ganglion cells. The physiological relevance of this effect is completely unknown. The patho-physiological relevance is highlighted by the large number of studies that indicate the role of retinal RAS in systemically induced retinal degenerations. Acknowledgements The work was supported by a research grant of the Ernst and Berta Grimmke Stiftung, Düsseldorf Germany. References Balakumar, P., Jagadeesh, G., 2014. Structural determinants for binding, activation, and functional selectivity of the angiotensin AT1 receptor. J. Mol. Endocrinol. 53, R71–R92. Barro-Soria, R., Stindl, J., Muller, C., Foeckler, R., Todorov, V., Castrop, H., Strauss, O., 2012. Angiotensin-2-mediated Ca2+ signaling in the retinal pigment epithelium: role of angiotensin-receptor-associated-protein and TRPV2 channel. PLoS One 7, e49624. Berka, J.L., Stubbs, A.J., Wang, D.Z., DiNicolantonio, R., Alcorn, D., Campbell, D.J., Skinner, S.L., 1995. Renin-containing Muller cells of the retina display endocrine features. Investig. Ophthalmol. Vis. Sci. 36, 1450–1458. Bok, D., 1993. The retinal pigment epithelium: a versatile partner in vision. J. Cell Sci. Suppl. 17, 189–195. Brandt, C.R., Pumfery, A.M., Micales, B., Bindley, C.D., Lyons, G.E., Sramek, S.J., Wallow, I.H., 1994. Renin mRNA is synthesized locally in rat ocular tissues. Curr. Eye Res. 13, 755–763. Danser, A.H., Derkx, F.H., Admiraal, P.J., Deinum, J., de Jong, P.T., Schalekamp, M.A., 1994. Angiotensin levels in the eye. Investig. Ophthalmol. Vis. Sci. 35, 1008–1018. Datum, K.H., Zrenner, E., 1991. Angiotensin-like immunoreactive cells in the chicken retina. Exp. Eye Res. 53, 157–165. Deinum, J., Derkx, F.H., Danser, A.H., Schalekamp, M.A., 1989. Renin in the bovine eye. J. Hypertens. Suppl. 7, S216–S217. Deinum, J., Derkx, F.H., Danser, A.H., Schalekamp, M.A., 1990. Identification and quantification of renin and prorenin in the bovine eye. Endocrinology 126, 1673–1682. Downie, L.E., Vessey, K., Miller, A., Ward, M.M., Pianta, M.J., Vingrys, A.J., WilkinsonBerka, J.L., Fletcher, E.L., 2009. Neuronal and glial cell expression of angiotensin II type 1 (AT1) and type 2 (AT2) receptors in the rat retina. Neuroscience 161, 195–213. Edelman, J.L., Miller, S.S., 1991. Epinephrine stimulates fluid absorption across bovine retinal pigment epithelium. Investig. Ophthalmol. Vis. Sci. 32, 3033–3040. Ferrari-Dileo, G., Davis, E.B., Anderson, D.R., 1987. Angiotensin binding sites in bovine and human retinal blood vessels. Investig. Ophthalmol. Vis. Sci. 28, 1747–1751. Fletcher, E.L., Phipps, J.A., Ward, M.M., Vessey, K.A., Wilkinson-Berka, J.L., 2010. The renin-angiotensin system in retinal health and disease: its influence on neurons, glia and the vasculature. Prog. Retin. Eye Res. 29, 284–311. Fukuda, K., Hirooka, K., Mizote, M., Nakamura, T., Itano, T., Shiraga, F., 2010. Neuroprotection against retinal ischemia-reperfusion injury by blocking the angiotensin II type 1 receptor. Investig. Ophthalmol. Vis. Sci. 51, 3629–3638. Guenther, E., Hewig, B., Zrenner, E., Kohler, K., 1997. Angiotensin II-induced inhibition and facilitation of calcium current subtypes in rat retinal ganglion cells. Neurosci. Lett. 231, 71–74. Guenther, E., Schmid, S., Hewig, B., Kohler, K., 1996. Two-fold effect of Angiotensin II on voltage-dependent calcium currents in rat retinal ganglion cells. Brain Res. 718, 112–116. Harrison, J.M., O'Connor, P.S., Young, R.S., Kincaid, M., Bentley, R., 1987. The pattern ERG in man following surgical resection of the optic nerve. Investig. Ophthalmol. Vis.
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