Elsevier

Experimental Eye Research

Volume 91, Issue 5, November 2010, Pages 700-709
Experimental Eye Research

Nitric oxide amplifies the rat electroretinogram

https://doi.org/10.1016/j.exer.2010.08.014Get rights and content

Abstract

It is well established that nitric oxide (NO) participates in retinal signal processing through stimulation of its receptor enzyme, soluble guanylyl cyclase (sGC). However, under pathological conditions such as uveoretinitis, diabetic or ischemic retinopathy, elevated NO concentrations may cause protein S-nitrosation and peroxynitrite formation in the retina, promoting cellular injury and apoptosis. Previous electroretinogram (ERG) studies demonstrated deleterious effects of NO on the retinal light response, but showed no evidence for a role in normal signal processing. To better understand the function of NO in ocular physiology, we investigated the effects of exogenous NO, produced by NO donors with different release kinetics, on the flash ERG of the rat. Within a limited concentration range, NO strongly amplified ERG a- and b-waves, oscillatory potentials, and the scotopic threshold response. Amplification exceeded 100% under dark adaptation, whereas the photopic ERG and the isolated cone response were increased by less than 50%. Blocking photoreceptor-bipolar cell synapses by AP-4 demonstrated a significant increase of the isolated a-wave by NO, and modeling the ERG generator PIII supported photoreceptors as primary NO targets. The sGC inhibitors ODQ and NS2028 did not reduce NO-dependent ERG amplification, ruling out an involvement of the classical NO effector cyclic GMP. Using immunohistochemistry, we show that illumination and exogenous NO altered the S-nitrosation level of the photoreceptor layer, suggesting that direct protein modifications caused by elevated levels of NO may be responsible for the observed phenomenon.

Research highlights

► NO strongly amplified the ERG. ► Photoreceptors were shown to be primary NO targets. ► Effect was independent from cGMP. ► Mechanism may depend on S-nitrosation of photoreceptor proteins.

Introduction

Nitric oxide (NO) is an intercellular signaling molecule produced by the three isoforms of NO synthase (NOS), the constitutive calcium-dependent neuronal and endothelial NOS (nNOS and eNOS) and the inducible, calcium-independent isoform (iNOS) (Alderton et al., 2001). In the nervous system, normal physiological concentrations of NO are estimated to be in the low nanomolar range (Garthwaite, 2008). At these concentrations, NO acts through activation of its specific receptor soluble guanylyl cyclase (sGC), elevating intracellular 3′,5′-cyclic guanosine monophosphate (cGMP). Alternatively, NO may posttranslationally modify proteins through S-nitrosation (also called S-nitrosylation) of specific cysteine residues (Foster et al., 2003, Jaffrey et al., 2001). Higher, sub-micromolar concentrations of NO can be reached under certain pathological conditions, mainly through expression of iNOS in microglia and other cells of the immune system (Duport and Garthwaite, 2005, Murphy and Gibson, 2007), or by a concerted activation of a large number of constitutive NOS enzymes within a limited space, as may occur in response to excitotoxic glutamate release caused by ischemia/reperfusion injury (Sattler et al., 1999). Such elevated concentrations of NO have the potential to damage cells and tissues by processes that are not yet completely understood. Candidate mechanisms include unspecific covalent modifications of proteins, lipids and DNA by reactive nitrogen species like peroxynitrite (“nitrosative stress”), and the inhibition of mitochondrial respiration by competition of NO with O2 (Keynes and Garthwaite, 2004).

In the eye, pathologic conditions such as diabetic retinopathy or glaucoma may lead to upregulation of iNOS expression, possibly resulting in NO levels that far exceed normal concentrations (Goldstein et al., 1996, Parikh et al., 2008, Toda and Nakanishi-Toda, 2007, Zhang et al., 1999, Zheng et al., 2007). This hypothesis is supported by the observation that decreasing endogenous NO levels through NOS inhibition prevents white light-induced retinal degeneration and cone cell death in a model of retinitis pigmentosa (Donovan et al., 2001, Komeima et al., 2008), while exogenous NO was shown to cause photoreceptor damage and apoptosis, retinal thinning and a significant reduction of response amplitudes measured by flash electroretinography (Fawcett and Osborne, 2007, Takahata et al., 2003a, Takahata et al., 2003b, Zoche and Koch, 1995). However, a limitation of many studies involving NO donors is that the applied NO concentrations are not quantified, and may easily exceed maximum endogenous levels by several orders of magnitude.

On the other hand, abundant evidence supports a physiological function of NO in retinal light responses. It is well established that NO synthesized by neuronal NOS (nNOS) in amacrine and ganglion cells participates in visual signal processing in the inner retina (Ding and Weinberg, 2007, Hoffpauir et al., 2006, Mills and Massey, 1995, Wang et al., 2003). Several studies also reported expression of nNOS in photoreceptor inner segments (Haberecht et al., 1998, Koch et al., 1994, Neufeld et al., 2000, Steinle et al., 2009), which could modulate phototransduction and shape the signals transmitted to bipolar and horizontal cells (Gotzes et al., 1998, Kourennyi et al., 2004, Levy et al., 2004, Savchenko et al., 1997, Snellman and Nawy, 2004). As cytosolic calcium levels are elevated in photoreceptors under darkness, calcium-dependent NOS isoforms should be activated under this condition, suggesting that NO constitutes a signal promoting dark adaptation. Several observations support this hypothesis. Exogenous NO increased the sensitivity of isolated tiger salamander rods to potassium-depolarization (Kourennyi et al., 2004), elevated light responses in rabbit horizontal cells (Xin and Bloomfield, 2000), and potentiated bipolar cell responses to simulated light flashes in mice (Snellman and Nawy, 2004). In addition, nNOS knockout mice showed reduced retinal sensitivity to light (Wang et al., 2007). Other studies reported an amplification of cone-dependent signals by NO, suggesting that NO contributes to light adaptation instead (Levy et al., 2004, Sato and Ohtsuka, 2010).

The aim of our investigation was to reassess the effect of exogenous NO on the retina of live rats and to distinguish between concentration-dependent physiological and noxious NO effects. Surprisingly, limited concentrations of NO significantly amplified the ERG signal amplitude, depending on the degree of light/dark adaptation, through a cGMP and cAMP independent mechanism. Our results further suggest that intraocular NO from endogenous or exogenous sources may cause nitrosative protein modifications, which is discussed in the context of current concepts of NO signaling under physiological and pathological conditions.

Section snippets

Materials and methods

All experiments were performed on 3–4 week-old Sprague Dawley rats irrespective of sex or weight. The rats, born and raised in the animal facility of the University of Valparaiso, were held at 20–30 °C under a 12 h photoperiod with water and food ad libitum. The experimental procedures were approved by the bioethics committee of the University of Valparaiso, in accordance with the bioethics regulation of the Chilean Research Council (CONICYT), and complied with the ARVO Statement for the Use of

ERG recordings

Here we show that exogenous NO, produced by NO donors injected into the vitreous of deeply anesthetized rats, strongly amplified the flash ERG of the rat (Fig. 1A–D). The effect was evident in the ERG a- and b-waves, OPs and STRs, which are thought to reflect photoreceptor, bipolar cell, amacrine cell and ganglion cell signaling, respectively (Bui and Fortune, 2004, Weymouth and Vingrys, 2008). Mathematical analysis of the ERG generators underlying the a- and b-waves, PIII and PII, revealed an

Discussion

The present study demonstrates that exogenous NO dramatically increased the amplitudes of all components of the scotopic flash ERG. Blockade of synaptic transmission to ON bipolar cells did not abolish NO-induced amplification of the ERG a-wave, supporting photoreceptors as site of NO action, although an independent effect on bipolar and other retinal cells cannot be excluded (Shiells and Falk, 1992). Both the cone response isolated by a twin-flash protocol, and the photopic ERG also showed a

Acknowledgements

This study was supported by the Chilean government through FONDECYT grant No. 1090343 and PBCT-CONICYT projects ACT-45 and RED-24.

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