Calcium (Ca²⁺) is an essential ion that is involved in the regulation of several processes in the body such as signal transduction pathways, contraction, secretion, blood coagulation, gene expression, apoptosis, necrosis, cell division, and endocytocis (Williams, 1974; Shuttleworth, 1997; Berridge, 2005; Carafoli, 2005; Wimmers et al., 2007). Within the cell, free intracellular [Ca²⁺]_I content is tightly regulated at about 100 nM to maintain a steep inwardly directed concentration and electrochemical gradients across the cell membrane by an interplay of several Ca²⁺ channels, pumps, transporters, buffering systems and intracellular storage organelles (Bogeski et al., 2011). Several ion channels facilitate transmission of [Ca²⁺] ions across the membranes: the voltage-gated calcium channels (CaV), transient receptor potential (TRP) ion channels, transmitter-gated Ca²⁺ permeant ion channels and the store operated Ca²⁺ entry (SOCE) and Ca²⁺ released-activated Ca²⁺ (CRAC/Orai) channels (Bogeski et al., 2011). Although various potential molecular targets for calcium channels have been identified, only the L-type voltage-activated calcium channels have found wide therapeutic beneficial application. There is evidence in the eye for the existence of a calcium transport system. Ca²⁺ has been reported to play a key role in mammalian lens physiology and pathology. Excessive levels of Ca²⁺ have been implicated in cortical cataract and there is presence of Ca²⁺ linked receptors in the lens (Rhodes and Sanderson, 2009). Voltage-gated Ca²⁺ channels: transient (T-type) and dihydropyridine-sensitive long-lasting (L-type) channels, have been reported to be expressed in muller cells of the retina (Puro and Mano, 1991; Puro et al., 1996; Bringmann et al., 2000). The retinal pigment epithelium (RPE) has also been reported to expresses voltage- and ligand-gated Ca²⁺conducting channels (Wimmers et al., 2007). These channels act as regulators of secretory activity, and thus contribute to RPE function. Changes in Ca²⁺ channel function, or activity has been shown to lead to degenerative diseases of the retina (Wimmers et al., 2007).

Despite the implication of Ca²⁺ in ocular physiology and pathology, there is a great need for studies centered on the regulatory role of H₂S and its interaction with calcium channels. Only one study to date, has addressed the possible interaction of H₂S and Ca²⁺ channels in the eye. In this study the authors' report that the production of H₂S in retinal neurons is regulated by intracellular Ca²⁺, (Mikami et al., 2011) and in turn H₂S can suppress Ca²⁺ channels by activating vacuolar type H⁺-ATPase (V-ATPase). Furthermore, the study also demonstrated that H₂S can suppress the elevation of Ca²⁺ in photoreceptor cells by activating V-ATPase in horizontal cells and thus maintain Ca²⁺ homeostasis. From these observations, the authors conclude that H₂S protects photoreceptor cells from the insult caused by excessive levels of light. Clearly results from this study provides a new insight into the regulation of H₂S production and the modulatory interaction of H₂S and Ca²⁺ channels in retinal transmission. In addition, the study postulates a cytoprotective effect of H₂S on retinal neurons and provides a basis for the therapeutic target for retinal degeneration. Increasing knowledge about the properties of Ca²⁺channels in ocular tissuess especially the retina will not only provide a new understanding of ocular function but could also provide a better understanding of the role of H₂S in ocular health and vision.

Potassium ion (K⁺) channel family represents one of the most prominent and ubiquitous ion channels in living organisms where their physiological role range from regulation of the action potentials in excitable cells to regulation of transepithelial transport processes, intracellular pH, cell survival and growth factor secretion in non-excitable cells (Ashcroft and Gribble, 1999; Bauer et al., 1999; MacDonald and Wheeler, 2003; Warth, 2003; Masi et al., 2005). K⁺ channel family consists of four subfamilies, the inwardly rectifying K⁺ (K_ir)-channels, voltage-gated K⁺ channels, Ca²⁺-activated K⁺ channels, and two-pore or leak K⁺-channels that are classified based upon number of transmembrane domains and electrophysiological properties. In the eye, K⁺ channels play central roles in maintaining ion, fluid balance and membrane potential. Several K⁺ channel subtypes such as voltage-gated K⁺ (Kv) channels and 4-aminopyridine (4-AP)-sensitive K⁺ channels are expressed in mammalian corneal epithelial cells (Rae, 1985; Rae et al., 1990; Rae and Farrugia, 1992). Studies have shown that changes in K⁺ channel activity modulate essential corneal epithelial functions needed for tissue homeostasis (Wolosin and Candia, 1987; Klyce and Wong, 1977). Furthermore, emerging evidence suggest that K⁺ channels play a crucial role in controlling apoptosis and proliferation in corneal epithelial cells (Lu et al., 2003; Roderick et al., 2003). Three major potassium currents (an outwardly rectifying current, an inwardly rectifying current, and a calcium-activated current) have been characterized in several mammalian lens epithelial cells (Rae, 1986; Cooper et al., 1991). These potassium conductances are essential for the maintenance of lens volume and transparency. Inwardly rectifying potassium (Kir) channel was reported to be highly expressed in bovine and human trabecular meshwork cells, (Llobet et al., 2001) as well as muller glial cells of the retina (Kofuji et al., 2002). K⁺ channels (Kv11; ether à-go-go related gene; erg) belonging to the family of voltage-gated K⁺ channels are present in mouse and human retina with the most abundant expression in rod bipolar cells. These channels are also found in the inner and outer plexiform layer, inner segments of photoreceptors, as well as the retina pigment epithelium (Cordeiro et al., 2011). These channels are vital for the control of the membrane potential in retinal neurons. Given the importance of K⁺ channel modulation in ocular tissues, evidence of an interaction between H₂S and these channels in the eye is imperative for understanding the role of H₂S as a signaling molecule in ocular functions.

The pharmacological effects of H₂S in the vasculature and brain, has been reported to involve K⁺ channels. To the best of our knowledge there are no studies in the literature pertaining to the effects of H₂S on K⁺ channels in the eye, except for those generated from our laboratory. In previous studies we have demonstrated that the pharmacological effects of H₂S (using H₂S–releasing compounds) in ocular tissues are partly mediated by K_ATP channels (Monjok et al., 2008; Kulkarni et al., 2009; Opere et al., 2009; Ohia et al., 2010). With the use of specific channel blockers, we report that H₂S interacts with K_ATP channels to relax ocular smooth muscle, and alter sympathetic and glutamergic neurotransmission in the anterior uvea and retina (Monjok et al., 2008; Kulkarni et al., 2009; Opere et al., 2009; Ohia et al., 2010). Furthermore, we recently show that K_ATP channels are involved in the regulatory role of H₂S in signal transduction processes in retina pigment epithelium cells (Njie-Mbye et al., 2012). It is reported that K⁺ channels play vital roles in cellular functions including vascular tone regulation, mediating neurotransmitter release, and neuroprotection in cardiovascular and CNSs (Yamada and Inagaki, 2005). Thus it is tempting to speculate a physiological role of H₂S in ocular tissues that involves the activation of K⁺ channels.

Chloride (Cl⁻) is one of the most prominent anions in the body that is involved in the regulation of a variety of important physiological and cellular functions such as volume homeostasis, organic solute transport, cell migration, cell proliferation, cell differentiation, and apoptosis. Unlike most physiological ions whose levels are tightly regulated within a limited range, the resting Cl⁻ ion concentration varies in different mammalian cell types and in developing cells (Wimmers et al., 2007). Cl⁻ conductance across membranes is facilitated by several pumps and co-transporters that are localized in plasma membranes and membranes of intracellular organelles. For example, chloride influx is facilitated by the Na⁺/K⁺/Cl⁻ co-transporters, Cl⁻/HCO₃-exchangers, and Na⁺/-Cl⁻ co-transporters while efflux is achieved by the cell K⁺/-Cl⁻co-transporters and Na⁺-dependent Cl⁻/HCO₃ – exchanger. Other channels and transporters expressed in intracellular membranes as well as Cl⁻ -binding proteins regulate intra-vesicular pH and Cl⁻ concentration (Duran et al., 2010). Several channels mediate passive flow of Cl⁻ ions across membranes. With exception of the transmitter-gated GABA and glycine receptors, these Cl⁻ channels are broadly classified into five subfamilies, the voltage-sensitive ClC subfamily, calcium-activated channels, high- (maxi) conductance channels, the cystic fibrosis transmembrane conductance regulator (CFTR) and volume-regulated channels (Verkman and Galietta, 2009). So far, only the voltage-sensitive ClC subfamily, CFTR and the transmitter-gated channels have been well described. In general, Cl⁻ channels are fairly non-selective for inorganic ions. Dysfunctional Cl⁻ channels have been linked to channelopathies such as myotonia congenita and cystic fibrocis (Duran et al., 2010). Cl⁻ channels are more abundantly expressed in the anterior segment of the eye due to Cl⁻ being the principal anion of aqueous humor secretion. Studies show that chloride efflux plays an important role in aqueous humor production and chloride channels present in the ocular ciliary epithelium are involved in aqueous humor homeostasis. Chloride currents have been reported to be present in bovine non-pigmented ciliary epithelium (NPE) and in transformed cultured human NPE (Chen and Sears, 1997). High-(maxi) conductance chloride channels are expressed in ciliary pigmented epithelial (PE) cells, (Do et al., 2004) whilst cAMP-activated Cl⁻ channels are present in the basolateral membrane of nonpigmented (NPE) ciliary epithelium (Edelman et al., 1995). CFTR is functionally expressed in corneal and conjunctival epithelium, corneal endothelium, and RPE (Shiue et al., 2002; Sun and Bonanno, 2002; Turner et al., 2002; Blaug et al., 2003; Levin and Verkman, 2005; Reigada and Mitchell, 2005). CFTR expression patterns in these tissues suggest the involvement of these chloride channels in regulation of tear film volume, corneal hydration and transparency, aqueous humor volume and IOP, and subretinal compartment size and ionic composition. In the retina, several Cl⁻ transporter and channels including the Na⁺/K⁺/Cl⁻ cotransporters, CFTR, and the voltage-sensitive ClC subfamily were reported to be highly expressed in the pigment epithelial layer (Zhang et al., 2011).

The activation of Cl⁻ channels by H₂S in the CNS has been shown as a protective mechanism for neurons from oxytosis (Tang et al., 2010). Electrophysiological evidence demonstrates that H₂S interacts with Cl- channels in the vasculature (Tang et al., 2010). To the best of our knowledge there are no studies reporting the interaction of H₂S with Cl⁻ channels in the eye. The observation of the presence of chloride channels in ocular tissues especially in the anterior uvea, coupled with evidence of channel activation by H₂S in non-ocular tissues, suggest possible regulation of chloride fluxes by H₂S in the eye with neuroprotective consequences and IOP lowering effects.

Glutamate is the major excitatory neurotransmitter in the mammalian CNS. Under normal physiological conditions, extracellular glutamate is tightly regulated (resting levels ≤1 μM) by five distinct excitatory amino acid transporters, EAAT1 (glutamate/aspartate transporter [GLAST]); EAAT2 (glutamate carrier [GLT-1]), EAAT3 (excitatory amino acid carrier 1 [EAAC1]), EAAT4 and EAAT5 (Zerangue and Kavanaugh, 1996; Levy et al., 1998). Excessive extracellular glutamate is known to lead to excitotoxicty in neuronal tissues. Thus, EAAT transporters play the essential role of rapidly terminating synaptic transmission, maintaining low ambient extracellular glutamate while simultaneously conserving neuronal glutamate for reuse via the glutamate-glutamine cycling system (Copenhagen et al., 2002; Zou and Crews, 2005). Glutamate transporter uptake activity is accompanied by a net inward movement of positive ions (3Na⁺:1H⁺ co-transport versus K⁺ counter-transport) (Kanner, 2006) and could be coupled to Na, K-ATPase pump (Rose et al., 2009). Glutamate transporters exhibit differential distribution in different tissues. In the mammalian retina, using immunocytochemical studies, EAAT1 (GLAST) has been shown to be localized in Muller cells (Rauen et al., 1996; Pow and Barnett, 1999). EAAT2 have been identified in cone photoreceptor and bipolar cells (Rauen and Kanner, 1994) while EAAT3 (EAAC1) in inner retinal neurons (Rauen et al., 1996). EAAT4 is localized in retinal astrocytes (Ward et al., 2004) and EAAT5 in photoreceptors, bipolar cells and in some muller cells (Arriza et al., 1997; Pow and Barnett, 2000). Interestingly, EAAT5 is found exclusively in retina (Pow and Barnett, 2000). The sodium-dependent glutamate–aspartate transporter (GLAST or EAAT1) is the major glutamate transporter in muller cells. This glutamate transporter (EAAT1/GLAST) maintains extracellular glutamate at a low level to ensure a high signal-to-noise ratio for glutamatergic neurotransmission and thus shield neurons from excitotoxic damage. To the best of our knowledge there are no studies till date that have examined the effect of H₂S on glutamate transporter in ocular tissues. Only one study, by our laboratory has demonstrated that H₂S donors caused an attenuation of glutamatergic transmission in mammalian retinae (Opere et al., 2009). Although the exact mechanism of action is not clear,it is feasible that H₂S donors could decrease glutamatergic transmission in mammalian retinae due to involvement of EAAT.

The cystine/glutamate antiporter (System x⁻_C) is responsible for the Na⁺ independent electroneutral exchange of cystine and glutamate. It is a member of the heteromeric amino acid transporter family which is composed of a heavy subunit and a corresponding light subunit linked by a disulfide bridge (Lim and Donaldson, 2011). System x⁻_C is regulated by extra- and intracellular gradients of glutamate which drives the import of cystine coupled to export of glutamate (Fiorucci et al., 2006; Lowicka and Beltowski, 2007). System x⁻_C has two major functions. First, it mediates cellular uptake of cystine for the maintenance of intracellular levels of glutathione, essential for protection of cells from oxidative stress. Second, it is instrumental in maintaining the redox balance between extracellular cystine and cysteine (Lo et al., 2008). In the eye, system x⁻_C has been identified in veterbrate lens) (Lim et al., 2005) and different parts of the mamamlian retina including the retinal endothelial cells, outer plexiform of retina, muller cells, retinal pigment cells and retinal ganglion (Kato et al., 1993; Bridges et al., 2001; Hosoya et al., 2002; Tomi et al., 2002; Dun et al., 2006). Oxidative damage of proteins is believed to underlie several major eye diseases such as age related nuclear (ARN) cataract, age related macular degeneration (AMD) and diabetic retinopathy (Lo et al., 2008). GSH, a major and potent antioxidant in the cells may prevent or slow down the progression of such diseases by protecting the thiol groups of proteins and minimizing oxidation-induced protein aggregation formation. However, oxidative stress alters rate of conversion of cysteine to glutathione and leads to depletion of glutathione levels (Fiorucci et al., 2006). System x⁻_C plays an important role in maintaining elevated intracellular levels of glutathione and serves as a potential therapeutic target for a number of ocular diseases.

In the CNS, H₂S demonstrates cytoprotective effect by protecting neurons and astrocytes, major type of glial cells from oxidative stress (Kimura, 2011a,b). H₂S enhances the activity of system x⁻_C and thus significantly increases the transport of cystine into neurons to increase the levels of substrate cysteine, for glutathione synthesis. Even in the presence of glutamate, H₂S significantly reverses the inhibition of cystine transport by glutamate (Kimura and Kimura, 2004). Based on the conclusions from these studies (Kimura and Kimura, 2004; Kimura, 2011a,b) in CNS, it will be interesting to investigate the effect of H₂S on system x⁻_C transporter in astrocytes and muller cells in retina under oxidative stress. Since, muller cells are the primary sites of glutathione localization in the retina, and the retina is extremely vulnerable to oxidative stress, understanding the function of H₂S on system x⁻_C in muller cells could play a pivotal role in protecting the retina from a variety of retinal diseases, such as diabetic retinopathy, age-related macular degeneration, and glaucoma. In the eye, there is evidence from few studies that demonstrate an increase in GSH production, following application of H₂S releasing drugs such as ACS67, ACS1. Although the mechanism of action is not clear, the authors suggest that intracellular cysteine levels are enhanced indirectly to form GSH by H₂S stimulation of glutamate/cystine antiporters (Sparatore et al., 2009; Perrino et al., 2009; Osborne et al., 2010). Moreover, as mentioned above, a study by Opere and Ohia (1997) had demonstrated the inhibitory action of H₂S donors on glutamatergic transmission in mammalian retinae. So, it is plausible that H₂S donors can render there cytoprotective effects by upregulating system x⁻_C transporter in the retina and thereby increasing the production of glutathione. As the exact mechanism of action needs to be investigated, there is very demanding need to understand the potential role of H₂S on the system x⁻_C transporter in the eye.

Cysteine transporters are widely distributed in various cell types including the muller cells of retina. Cysteine transporters readily import cysteine into the cell for direct conversion to glutathione (Bringmann et al., 2009; Mathai et al., 2009; Kimura, 2010). So far only one study by Kimura (2010) on brain cortex has shown the effect of H₂S on cysteine transporters. The study demonstrates that H₂S (acting as a reducing agent) reduces cystine into cysteine in the extracellular space and makes cells efficiently transport cysteine into cells for GSH production. Moreover authors state that, the contribution of cysteine transport to the production of GSH is much greater than that of cystine transport. This production of GSH is further enhanced by H₂S under conditions of oxidative stress caused by excessive glutamate toxicity. To the best of our knowledge there is a lack of studies on cysteine transporters in the eye and no evidence of the effect of H₂S on these transporters in ocular tissues. Although the production of H₂S in the eye is not well understood as there is only one study, from our laboratory showing that this gasotransmitter is endogenously produced in ocular tissues (Kulkarni et al., 2011), it is possible that the release of H₂S may regulate the transport of cysteine in ocular tissues, thus facilitating the production of GSH. This increase in the levels of GSH by H₂S may contribute to the potential protective effect of H₂S.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.