A.Expression in the Human CB of Glutamate Transporters of the Excitatory Amino Acid Transporters Family

In the retina L glutamate is released by photoreceptors, bipolar cells, and retinal ganglion cells (Lam, 1997; Thoreson and Witkovsky, 1999). Whether L glutamate in the AqH derives from the retina is presently unknown. However, based on preliminary studies, it is not premature to speculate whether a putative glutamatergic system is expressed in the inflow–outflow pathways of AqH. Glutamate transporters, members of the excitatory amino acid transporters (EAAT) family, mediate the uptake of glutamate and exhibit currents that depend on extracellular Naþ. Studies suggest that this complex process also involves the cotransport of protons (Hþ) and gates the passive flux of Cl– ions (Arriza et al., 1997).

Screening of a human CB cDNA library, by PCR, with specific sets of oligonucleotide primers spanning coding regions of genes encoding distinct members of the EAAT family, has revealed the surprising amplification of EAAT1 and EAAT5 sequences. The expression of EAAT5 in the human CB was further examined by Northern blot, and the mRNA (3.1 kb) size of EAAT5 in this tissue was identical to the one described in the human retina (Arriza et al., 1997; Ghosh and Coca Prados, unpublished results). In the rat, rabbit, and monkey retinas, EAAT5 is restricted to rod photoreceptor cells (Pow and Barnett, 2000), in cone and rod photoreceptor terminals, and in axon terminals of rod bipolar cells in the mouse retina (Wersinger et al., 2006). The large Cl– conductance of EAAT5 suggests that it functions more like a ligand gated Cl– channel than a glutamate transporter. Cl– is mainly translocated in the presence of glutamate or related substrates, and Cl– movement is not thermodynamically coupled to substrate transport.

In certain forms of glaucoma, the expression of the glutamate transporter, EAAT1 (GLAST) has been shown to be expressed at reduced levels (Naskar et al., 2000) and suggested to be associated with retinal ganglion cell death (Vorwerk et al., 2000). Whether the glutamate receptors and glutamate transporters expressed in the CB are functional or play a role in disease, awaits further studies at the cellular, molecular, and electrophysiological levels. Finally, two enzymes known to be associated with the glutamate cycle were also detected in the human CB and TM cells. Of these enzymes, glutamine synthase, involved in the conversion of glutamate into glutamine, was described to be present in the NPE of the CE, and suggested to be involved in the synthesis of multipolysaccharides (Riepe and Norenberg, 1978). The second glutamate enzyme in the CB is glutaminase that converts glutamine into glutamate.

IX. IMPLICATIONS OF A NEUROENDOCRINE SIGNALING IN THE ANTERIOR SEGMENT OF THE EYE

A.Potential Neuroendocrine Entrainment of Circadian Rhythms: AqH Secretion and IOP

There is abundant evidence indicating that in the mammalian eye, AqH flow (Ericson, 1958; Reiss et al., 1984; Topper and Brubaker, 1985; Smith and Gregory, 1989; Maus et al., 1994), and IOP (Liu, 2001) exhibit well defined diurnal rhythms. The rate of secretion of AqH in humans falls 50– 60% during sleep (Brubaker, 1998), and IOP is higher during the night than during the day (Liu, 2001), suggesting distinct circadian signals modulating inflow and outflow during the day and during the night. Clarifying the mechanisms and regulation of circadian AqH secretion can lead to novel strategies for lowering IOP, the only known intervention in slowing the onset and progression of blindness in glaucoma. There is supporting evidence that factors found in AqH follow a circadian rhythm (Nii et al., 2001; Liu, 2000, 2002). Although these factors remain to be determined, other factors known to be present in the AqH and in the CE are presumably circadian (Fig. 6). Among these factors are melatonin, and the melatonin rhythm generating enzymes, arylalkylamine N acetylytransferase (AA NAT), and hydroxyindole O methyltransferase (HIOMT) (Martin et al., 1992). Melatonin has been demonstrated to be circadian in the tissues where it is synthesized including the pineal gland and retina. A number of additional endocrine factors in the AqH are predictably circadian including SST and cortisol, both synthesized and secreted by the CE (Rauz et al., 2003; Ghosh et al., 2006). SST, for example, has been shown to be circadian in the cerebrospinal fluid and chemically similar to the AqH (Berelowitz et al., 1981). In contrast, cortisol is the main circulating human glucocorticoid in plasma, exhibiting a diurnal rhythm, and a potent natural immunosuppressant activity. It has been suggested that cortisol may impose diurnal variation on immune responsiveness, similar to melatonin.

The circadian rhythmicity of AqH flow and IOP has been suggested to be synchronized by the master pacemaker (clock), the suprachiasmatic nucleus (SCN). The central clock in the SCN receives photic information from photoreceptor cells in the retinal inner layer through the retinohypothalamic tract (RHT) in the form of neural signals (the so called input signals). The input signals (i.e., glutamate, nitric oxide) turn on the circadian clock genes and reset the clock (Ding et al., 1997). The clock genes turn on the expression of other genes, the so called clock controlled genes, encoding output signals. Among the circadian output signals figure: endocrine/paracrine factors,

norepinephrine (NE), cAMP, arginine vasopressin, adenosine, and dopamine. These humoral and neural signals then synchronize circadian rhythm activities in peripheral tissues expressing circadian oscillators. In the adrenal gland, numerous genes, and entire pathways associated to catecholamine and steroid metabolism are transcriptionally regulated by an endogenous circadian clock in this tissue (Oster et al., 2006). In recent years, the existence of circadian clocks in peripheral tissues (Zylka et al., 1998; Balsalobre, 2002), and in dissociated cells (Balsalobre et al., 1998), has been demonstrated. These peripheral clocks are self sustained and cell autonomous (Balsalobre et al., 1998; Yamazaki et al., 2000; Balsalobre, 2002). Most importantly, the circadian clock in SCN and in peripheral tissues share the same molecular components. However, there is a significant diVerence between oscillations in peripheral tissues, and in cells in vitro which damped after time, and those recorded from SCN neurons which maintain circadian rhythmicity for a longer time.

It has been suggested that the human CB contains a peripheral clock involved in circadian rhythm of AqH secretion. This hypothesis is supported, in part, by the expression in the CB of the clock genes: Period (Per), Clock (Clock), Cryptochrome (Cry), BMAL 1, and Timeless (Tim). In peripheral tissues, clock genes oscillate as in the SCN (Ripperger and Schibler, 2001); form feedback loops, resulting in oscillations of expression levels of clock, and clock controlled genes with 24 hour cycles (King and Takahashi, 2000). Disruption of clock genes (i.e., Cry1) aVects the diurnal rhythm of IOP (Maeda et al., 2006). Cultured human ciliary epithelial cells (NPE) entrained to a 24 hour light dark (LD) cycle (i.e., 12 hour L and 12 hour D) resets the circadian rhythm of the clock gene Per1 by the action of the input signal NE, and exhibit circadian expression of cAMP, SST mRNA, and AA NAT (unpublished results).

We also have hypothesized that the neuroendocrine functions of the CB are most likely linked to an endogenous peripheral circadian oscillator. The secretion of hormones and neuropeptides by the CE in the AqH are output signal candidates for clock controlled genes, as shown in many peripheral clocks (Hastings et al., 2003; Oster et al., 2006). These output signals can potentially regulate AqH flow and IOP. Although the SCN may be important in the synchronization of circadian physiology in peripheral tissues, it has been shown that the input of the SCN or of photoreceptor (rods and cones) cells of the retina are not essential for maintaining the circadian rhythm in peripheral clocks. For example, the circadian AA NAT expression and activity in the retina occur in the absence of SCN (Tosini and Menaker, 1998; Iuvone et al., 2005). These studies suggest a high degree of autonomy in peripheral clocks which run with some degree of independence from the SCN.

The local influence of peripheral clocks on circadian physiology has two clear advantages. First, the central clock would need to maintain and/or reset the peripheral clocks through a limited number of ligands. And second, the local control of circadian rhythmicity would allow it to adapt rapidly. It has been suggested that key circadian proteins determine circadian periodicity in gene expression and physiology for many organs (Reppert and Weaver, 2002), and up to 10% of genes in diVerent tissues are directly or indirectly regulated by the circadian clock system (Panda et al., 2002; Storch et al., 2002). Since diVerent tissues have characteristic sets of genes with clock regulated timing and amplitude of expression, it is likely that the circadian clock in the CB may be involved in the control of aqueous inflow and outflow with distinct signals.

The CB may be capable to autosustain circadian activity due to the expression of many components of phototransduction and of the retinoid cycle (Bertazolli Filho et al., 2001; Ghosh et al., 2004; Salvador Silva et al., 2005). Melatonin is viewed as a hormonal message (output signal) for the duration of the dark phase in circadian rhythm. Melatonin receptors are also functionally expressed in the CE (Osborne and Chidlow, 1994), and under 24 hour LD cycle, the AA NAT activity in CB explants is higher in the dark than in the light. Recent studies indicate that melatonin receptors (MT3) activation expressed in the CB could modulate IOP reduction (Serle et al., 2004). Table III summarizes several biological functions assigned to the human CE.

B. Neuroendocrine Immune Circuitry

Neuropeptides in the AqH may exhibit biological activities associated with a putative neuroendocrine immune circuit in the anterior chamber (Fig. 6). It has been well recognized that the anterior segment of the eye is an immune privileged site, and that antigens injected into the anterior chamber elicit deviant systemic immune responses that are devoid of immunogenic inflammation. This distinctive response known as anterior chamber associated immune deviation (ACAID) arises in part by the soluble immunosuppressive and anti inflammatory factors released by the surrounding tissues into the AqH. The AqH is able to suppress interferon g (INF g) production by eVector T cells, and neuropeptides present in the AqH including a MSH, CGRP, VIP, and SST, which are capable of suppressing pathogen induced inflammation in the anterior chamber of the eye. SST, for example, contributes to the immunosuppressive properties of the AqH by promoting the production of the potent immunosuppressive cytokine a MSH, and by inducing the activation of regulatory T cells (Taylor and Yee, 2003).

TABLE III

Biological Functions Assigned to the Human Ciliary Epithelium

Synthesis and secretion of plasma proteins, proteases, anti proteases, anti angiogenic, and growth factors.

Expression of neuropeptides, transmitters, peptide hormones, and cognate receptors.

Expression of immunosuppressive factors TGFb2, somatostatin, substance P

Steroidogenic: Steroid converting enzymes (17b HSD2, 4, 5 and 7; 11bHSD1)

Prostaglandins: Cyclooxygenase 2 (COX 2); prostaglandin D2 synthase

Expression of components of rod phototransduction including rhodopsin, rhodopsin kinase, arrestin

Expression of components of the visual cycle including CRALBP, IRBP, CRBP, 11 cis RDH, LRAT

Expression of glutamate metabolizing enzymes, glutamate receptors, and glutamate transporters

Circadian clock: Expression of melatonin rhythm generating enzymes AA NAT, HIOMT, melatonin receptors, and clock genes

Expression of glaucoma genes including MYOC, CYP1B1, OPTN

The presence of pro inflammatory (i.e., substance P and CGRP) and anti inflammatory (SST) neuropeptides in AqH indicates that there is a regulated balance between these factors to influence the ocular immune privileged microenviroment of the anterior chamber. TGFb2, a cytokine that promotes immune deviation and immunosuppressive activities in the anterior chamber has been detected in high levels in the AqH in approximately 50% of glaucoma patients with primary open angle glaucoma (POAG) (Picht et al., 2001).

Neuropeptides with immunosuppressive and anti inflammatory properties in the AqH may derive either from the autonomic and sensory systems and/ or from the ocular tissues in the anterior segment. The pigmented cells of the iris, the bilayered CE, and possibly the TM cells are capable of synthesizing and releasing pro inflammatory and immunosuppressive factors. As indicated earlier, SST expression has been shown in the iris and the ciliary processes, and SST receptors have been detected in the NPE cells of the CE (Ghosh et al., 2006) and in TM (Fig. 3). The human TM expresses VIP and SgII (Gonza´lez et al., 2000b). Immunoregulatory actions of VIP are mediated by VIP receptors that are expressed on NPE cells. In mice, VIP induces the production of interleukins and displays anti inflammatory actions. High level of TGFb2 mRNA is expressed in human ciliary processes and in cultured NPE cells (Escribano et al., 1994), and in the TM as well (unpublished results), suggesting that these tissues have the potential ability

to produce immunosuppressive factors into the AqH. However, recent studies have documented that T cells exposed to cultured PE cells of the iris CB were induced to secrete large amounts of active and latent TGF b2, a response that is mimicked by exposing T cells to AqH (Knisley et al., 1991). These observations indicate that TGF b released into the AqH could be from multiple sources, and confirm that cells from the CE exhibit immunomodulatory functions of their own, since they have the ability to synthesize, and release cytokine factors independent of the involvement of T cells. The finding that the CE and TM cells express cognate receptors for many of the neuropeptides and cytokine released in the AqH is not coincidental, and it suggests that these factors may act on autocrine and paracrine based mechanisms to promote immune privilege in the anterior chamber of the eye.

Finally, neuropeptides with immunosuppressive properties in AqH are potentially under a 24 hour LD circadian cycle. There is evidence that ambient light is an important factor in inducing the ocular immune privilege in the anterior chamber (Ferguson et al., 1988) and that green light (500– 510 nm) within the visual spectrum is the relevant photic energy which influences intraocular immune reactions (Ferguson et al., 1992). Injection of antigen into the anterior chamber of mice, raised in complete darkness from birth, failed to induce ACAID whereas mice raised in the same manner and exposed to ambient light for 48 hours restored the capacity of the eyes to support ACAID. Interestingly, severance of the optic nerve had no eVect on the ocular immune privilege of the anterior chamber, suggesting that the presence of photoreceptors in the anterior segment of the eye may be involved in the modulation ACAID. It has been suggested that circadian factors including melatonin, expressed and released by the CE in the AqH, might contribute to the light dependent modulation of ACAID. Thus, neuropeptides released by the CE could exhibit multiple endocrine functions, including communication with cells of the AqH outflow pathways and the immune system, to maintain the immune privilege condition of the anterior segment of the eye (Fig. 6). Finally, the release of peptides in AqH could follow a 24 hour LD circadian rhythm cycle as described for AqH secretion and IOP.

X. SUMMARY

This chapter introduces the ‘‘inflow outflow link hypothesis.’’ It proposes that inflow and outflow of AqH are linked by neuroendocrine factors released by the CE. The neuropeptides released by the CE and carried out by the AqH may represent a major amplification system which establishes communication with cognate receptors expressed in cells of the outflow