the human corneal endothelium: new insights into

ARTICLE IN PRESS

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doi:10.1016/j.pr

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Progress in Retinal and Eye Research 26 (2007) 359–378

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The human corneal endothelium: New insights into electrophysiologyand ion channels

Stefan Mergler�, Uwe Pleyer

Department of Ophthalmology, Charite—University Medicine Berlin, Campus Virchow-Clinic, Augustenburger Platz 1, 13353 Berlin, Germany

Abstract

The corneal endothelium is a monolayer that mediates the flux of solutes and water across the posterior corneal surface. Thereby, it

plays an essential role to maintain the transparency of the cornea. Unlike the epithelium, the human endothelium is an amitotic cell layer

with a critical cell density and the risk of corneal decompensation. The number of endothelial cells subsequently decreases with age.

Moreover, the endothelial cell loss is accelerated after various impairments such as surgical trauma (e.g. cataract extraction) and

following corneal transplantation. This cell loss is associated with programmed cell death (apoptosis) and changed ion channel activity.

However, little is known about the electrophysiology and ion channel expression (in particular Ca2+ channels) in corneal endothelial

cells. This article reviews our current knowledge about the electrophysiology of the corneal endothelium. It highlights ion channel

expression, which may have a major role in corneal cell physiology and pathological events. A better understanding of the

(electro)physiological function of the cornea may lead to the development of clinical relevant new therapeutic and preventive measures.

r 2007 Elsevier Ltd. All rights reserved.

Keywords: Endothelial barrier and pump; Ion channels; Calcium homeostasis; Apoptosis

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360

2. Background: basic physiology of the corneal endothelium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360

3. Ion channels in corneal endothelium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362

3.1. Voltage-gated ion channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363

3.1.1. Sodium channels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363

3.1.2. Potassium channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364

3.1.3. Chloride channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364

3.1.4. Calcium channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365

3.2. Transient receptor potential channels (TRPs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366

3.2.1. Calcium-permeable TRPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366

4. Pathophysiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367

4.1. Pump-leak mechanism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 368

4.2. Cell loss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369

4.3. Are ion channels involved in apoptosis? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 370

5. Clinical relevance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371

5.1. Endothelial cell preservation during corneal banking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372

5.2. Corneal endothelial cell transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372

e front matter r 2007 Elsevier Ltd. All rights reserved.

eteyeres.2007.02.001

: Transient receptor potential channels (TRPs); Intracellular Ca2+ concentration ([Ca2+]i)

ing author. Tel.: +4930 450 559648; fax: +4930 450 559948.

ess: [email protected] (S. Mergler).

www.elsevier.com/locate/prer

dx.doi.org/10.1016/j.preteyeres.2007.02.001

mailto:[email protected]

ARTICLE IN PRESSS. Mergler, U. Pleyer / Progress in Retinal and Eye Research 26 (2007) 359–378360

5.3. Optimising of rinse solutions for intraocular surgery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373

6. Summary, conclusions, and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374

1. Introduction

The most significant property of the cornea is itstransparency. Thereby, the corneal endothelium plays anessential role by maintaining the stromal hydrationthrough Na+–K+-activated adenosine tri-phosphatase(ATPase) present in the basolateral borders of cornealendothelial cells (Tervo and Palkama, 1975). These activepump mechanisms ensure that stromal hydration remainsconstant even if the intraocular pressure (IOP) is fluctuat-ing. Simultaneously, the permeability is ensured which isessential for the nutrition of the corneal tissue (McDonaldet al., 1987; Bourne, 1998; Bourne et al., 1999). The vitalityof the corneal endothelium has a crucial importance for itsbarrier and pump functions. In addition, corneal thicknessstrongly associates with endothelial barrier and pumpfunctions. Consequently, breakdown of one or both ofthese parameters leads to corneal oedema (McDonald et al.,1987). The corneal endothelium is a monolayer of cellsthat forms a hexagonal mosaic on Descemet’s membrane.However, there are also non-hexagonal cells in the cornealendothelium (Doughty, 1992, 1998). Subsequent studiesindicated that the mosaic is formed following a specificorder instead of a random layout (Doughty, 1998) (Fig. 1).

In contrast to the corneal endothelium of othervertebrates, human corneal endothelium does not containmitotically active cells (Joyce, 2003). As a consequence, thenumber of corneal endothelial cells in vivo decreases withthe human aging process (Laule et al., 1978; Li, 1985). Thecells become more flat with increased cell surface area.Recent studies prompted the hypothesis that, with aging,an increased number of human corneal endothelial cellsenters a replicative senescence-like state in which theybecome increasingly refractive to mitogenic stimulation(Joyce, 2003). In addition, there are various conditions thatcan increase or accelerate endothelial cell loss, such ascorneal endothelial dystrophy or increased IOP afterkeratoplasty (Gagnon et al., 1997; Reinhard et al., 2001,2002; Bohringer et al., 2001; Langenbucher et al., 2002;Bertelmann et al., 2004; Birnbaum et al., 2005). Thecorneal dystrophy-associated cell loss is often caused byapoptosis and changed ion channel activities (Cho et al.,1999; Krick et al., 2001; Li et al., 2001). However, little isknown about the mechanisms through which ion channels[e.g. voltage-operated calcium channels (VOCCs), transientreceptor potential channels (TRPs)] function in humancorneal endothelium. This lack of information hampers theunderstanding of both the role of ion channels in thehuman corneal endothelium and in various cornealdiseases. An increased knowledge in this field will provide

a better understanding of physiology and pathophysiologyof the cornea. Potentially, this may lead to the developmentof targeted new therapies against a reduction of cornealpump function capacity caused by corneal diseases,apoptosis or traumatic/iatrogenic procedures as well aspreventive measures to protect corneal endothelial cell loss.

2. Background: basic physiology of the corneal endothelium

Historically, first fundamental investigations about thecorneal structure and transparency were performed byMaurice in the 1950s (Maurice, 1957). He proposed a so-called lattice theory, which states that collagen fibrils are ofequal diameter (275–350 A) and are equally distant fromeach other (Fig. 2). In the stroma of a swollen cornea thathas lost its transparency, the distance between thesecollagen fibres within the lamellae is increased (Edelhauseret al., 1994). In further studies, the physiologic importanceof the corneal epithelial and endothelial barrier andmetabolic pump functions attract increased interests. Ionand fluid balance should be maintained in order todehydrate the cornea and to prevent corneal swelling.The dehydration process is important for maintainingcorneal transparency (Maurice, 1972). If either limitinglayer is compromised, the cornea will increase in thickness,become oedematous, and lose its transparency. The effectof epithelial removal and endothelial damage on cornealthickness was first investigated in the rabbit (Edelhauseret al., 1979, 1982; Cohen et al., 1979). Importantly, therewas noteworthy corneal swelling following endothelialdamage. A similar observation was subsequently made inisolated and perfused human corneas, showing that agreater degree of corneal swelling occured when theendothelial barrier was removed compared to the epithelialbarrier (Cristol et al., 1992).Corneas that have been stored in various preservation

media prior to keratoplasty have a minimal loss ofproteoglycans, indicating that the epithelial and endotheliallayers remain intact (Slack et al., 1992). However, there issubstantial proteoglycan loss from the cornea to thepreservation medium during storage if the corneal epithe-lium or endothelium or both are removed. Clinically, whenthere is an increase in corneal thickness (i.e. stromaloedema) associated with uptake of water, there is also anassociated loss of stromal proteoglycans. Thus, prior torestoring the cornea to a normal thickness, the epithelialand endothelial barriers should be re-established so thatthe metabolic pump can actively transport ions for keepingthe cornea relatively dehydrated.

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Fig. 1. Left: Typical light micrograph of the corneal endothelium of a pig (scale bar ¼ 120mm). Right: Drawing of a mosaic arrangement of central

corneal endothelial cells of a young Caucasian adult showing the 6-sided cells (scale bar ¼ 135mm). [From Doughty, Ophthalmic Physiol. Opt. 1998;

reprinted by permission from Elsevier Science Ltd. (The College of Optometristsr 1998).]

Fig. 2. Structures of the cornea and the sclera. The collagen fibrils of the

cornea (left) are illustrated as perfectly ordered (idealised lattice model).

[From Ameen et al., Biophys. J. 1998; reprinted by permission from

Biophysical Societyr 1998.]

S. Mergler, U. Pleyer / Progress in Retinal and Eye Research 26 (2007) 359–378 361

Based on the above-mentioned lattice theory and uniform

refractive index theory, the cornea maintains its transpar-ency (Churms, 1979; Ameen et al., 1998). In more recent

literature, the lattice theory is favoured to the refractiveindex theory (Fig. 2).Ameen used a method of photonic band structure to

calculate the frequencies of light that propagate in latticemodels of the cornea and sclera of the mammalian eye.Their calculations showed that the dispersion relation forthe cornea is linear in the visible light range, implying thatthe cornea is transparent (Ameen et al., 1998). In contrast,as the proteoglycans absorb water in the stroma of aswollen cornea that partially lost its transparency, thedistance between collagen fibres within the lamellae isincreased.The endothelial monolayer functions as a permeability

barrier that restricts the movement of water and solutesinto the hydrophilic stroma. For this function, an intactmonolayer of endothelial cells is essential. If the integrity ofthe monolayer is breached, corneal oedema rapidlydevelops (Nielsen et al., 1982). In contrast to the cornealepithelium, which forms a tight barrier, the endothelialbarrier is a permeable (leaky) barrier. In an intact cornea,aqueous humour does cross the endothelium and enters thestroma at a slow but constant rate. There is a constant leakof aqueous providing the major source of nutrients for cells

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Fig. 3. Electro-osmotic fluid flow: proposed scheme for electrical currents

and ionic fluxes across corneal endothelial cells. Ioc is the spontaneously

circulating open circuit current (Ioc ¼ 25 mA/cm2), originating from a

transendothelial potential difference of 500V and a paracellular resistance

Rp (�20O cm2). The return current goes through the cells. Fluxes are

depicted in open circuit (no currents imposed). Electro-osmotic coupling

would take place at negatively charged intercellular junctions, through

which current would be carried predominantly by the transjunctional

Na+ fluxes shown. [From Fischbarg, J. Exp. Zool. 2003’ reprinted by

permission from Wiley-Liss, Inc.r 2003.]

S. Mergler, U. Pleyer / Progress in Retinal and Eye Research 26 (2007) 359–378362

of the non-vascular cornea. This property is due to thepresence of intercellular gap junctions (GP) and tightjunctions (TJ) at the apical membrane of endothelial cells.However, the TJ have more importance as permeabilitybarriers (Watsky et al., 1990) and they do not provide acomplete seal around the cell. Therefore, aqueous humourcan leak into the paracellular space.

For the barrier and pump functions, vitality of thecorneal endothelium has fundamental importance. Fluidand solutes are continuously leaking into the stroma. Inaddition, there is endothelial net sodium and bicarbonateion transport (Huff and Green, 1981; Hodson and Miller,1976) from stroma to aqueous humour. The maintenanceof corneal thickness and transparency is based on equalvolume of such fluid leaking into the stroma and fluid thatactively removed from the stroma by the endothelium(Bourne, 1998). Bryant developed a model that relies on theDonnan view of corneal swelling as well as the ‘‘pump-leakhypothesis’’ (Bryant and McDonnell, 1998). In thiscontext, the Na+–K+-ATPase plays an important role(Bonanno, 2003). This integral membrane protein islocalised to the (baso-) lateral cell membrane in cornealendothelium (Stiemke et al., 1991; Guggenheim andHodson, 1994). Strong evidence indicates that this enzymeis an essential component of endothelial pump function.Further studies confirmed the importance of the Na+–K+-ATPase to endothelial net ion transport (Midelfart andRatkje, 1985; Riley et al., 1994). It appears to be directlyinvolved in the active transport of sodium across thecorneal endothelium. However, selective inhibition of thisenzyme by ouabain decreases endothelial active sodiumflux whereas TJ are not affected (McDonald et al., 1987;Watsky et al., 1990; Stiemke et al., 1991; Riley et al., 1997).In this context, Fischbarg presented evidence in cornealendothelium for electro-osmosis as the mechanismunderlying fluid transport. A local recirculating electriccurrent would result in electro-osmotic coupling at the levelof the intercellular junctions, dragging fluid via theparacellular route. He proposed a model for electricalcurrents and ionic fluxes across corneal endothelial cells(Fig. 3) (Fischbarg, 2003).

Electrical proceedings have also been detected at woundsin human skin and in rodent cornea and skin due tochanges of the electric fields (EF) (Zhao et al., 2006). EFare generated when the epithelial layer is cut and the lesionshort-circuits the transepithelial potential difference (Reidet al., 2005). In every species studied, disruption of anepithelial layer instantaneously generates endogenous EF,which have been proposed to be important in woundhealing (McCaig et al., 2005). Wound-induced EF werealso investigated in corneal epithelial cells. In addition,drugs were able to involve the effects on ion transport andwound healing (Reid et al., 2005). Reid and colleaguesapplied an EF, which was able to increase wound healing.In contrast, applying a field that has a polarity oppositefrom the normal wound field can reverse wound healing ina corneal epithelium tissue block preparation (Reid et al.,

2005). In this context, Asamoah et al. (2003) described afluorometric approach to local EF measurements involtage-gated ion channels.

3. Ion channels in corneal endothelium

Ion channels are transmembrane pores presentinghydrophilic channels for ions to cross a lipid bilayer downtheir electrochemical gradients. Stimulation by voltagechanges, neurotransmitters or mechanical stress opens aprotein pore across the plasma cell membrane throughwhich selected ions pass through. Ion channels mediaterapid (electrical) signalling events enabling sight, sensation,movement, and regulation of fluid and electrolyte home-ostasis. They play important roles in various physiologicalfunctions such as control of muscle contraction, volumeregulation, and hormone secretion. Approximate 300different types of ion channels are predicted in the humangenome (Nilius et al., 2005; Clapham et al., 2005; Kuboet al., 2005; Catterall et al., 2005a, b). Ion conductivities ofthese channels are usually observed and typically a millionions per second may flow. Ion channels may be voltage-gated, like sodium and potassium channels, or VOCCs.Furthermore, they also may be ligand-gated, like theacetylcholine receptor. One superfamily of ion channels,the transient receptor potential (TRP) superfamily functionis a field of current interest, especially in sensoryphysiology (Clapham, 2003; Montell, 2005). More recentstudies indicated that ion channels are widely regarded tobe causally involved in many diseases (so-called channelo-

pathies) or contributing indirectly to the genesis of severaldiseases (Nilius et al., 2005). However, ion channels havenot yet been systematically searched as candidates forchannelopathies. Only a few studies focused on ion channel

ARTICLE IN PRESSS. Mergler, U. Pleyer / Progress in Retinal and Eye Research 26 (2007) 359–378 363

types that are associated with human diseases and inparticular with cancer, but the number is these studies areconstantly increasing (Nilius et al., 2005; Schonherr, 2005).The electrophysiological characteristics of ion channels areinvestigated by patch-clamp techniques (Hamill et al.,1981; Horn and Marty, 1988). The measuring principle ofthe patch-clamp technique represents a particular voltage-clamp procedure (Fig. 4). Briefly, there are the whole-celland single-channel currents that will be measured andregistered in the voltage-clamp and patch-clamp experi-ments. Finally, identification of the origin of these currentsleads to insight about membrane conductance, ion channelidentity, ion selectivity, and other related phenomena.

The ‘‘whole-cell’’ mode of the patch-clamp allowsassessment of the macroscopic current from single cellsisolated from almost every tissue in the body. Generally,ion channels play essential roles in maintaining ion andfluid balance in order to prevent corneal swelling. How-ever, there are only a few studies about the mechanismsthrough which ion channels, especially Ca2+-permeableion channels, function in human corneal endothelium. Thisvoid leaves it unclear in the relationship among defectiveion channel control, cell death, and disease.

3.1. Voltage-gated ion channels

Voltage-dependent or voltage-gated ion channels aretransmembrane protein pores that are permeable to ions

Fig. 4. Simplified diagram of a patch-clamp amplifier and an equivalent circ

voltage, Vpip: pipette potential, Vout: output voltage proportional to the curr

converter, which is installed in a little box (preamplifier) near the pipette. After

Thereby, membrane fragments and other cell debris are sucked near the pipet

and particularly sensitive to the change of transmembranepotential difference. These channels are crucial forneuronal signal transmission and intracellular signaltransduction.

3.1.1. Sodium channels

Generally, the family of voltage-gated sodium (Na+)channels initiates action potentials in all types of excitablecells. The voltage-gated Na+ channel is a target of many ofthe deadliest neurotoxins such as tetrodotoxin (TTX)(Catterall et al., 2005a). So far, little is known aboutvoltage-dependent Na+ channels in non-excitable cellssuch as human corneal endothelial cells. Firstly, Watskyet al. (1991) detected voltage-sensitive TTX- and quinidine-blockable Na+ channels in the rabbit corneal endotheliumand the frog lens epithelium using patch-clamp techniques(Fig. 5).This is surprising because TTX-sensitive Na+ channels

are normally not found in non-excitable cells like cornealendothelial cells. However, they were already detected incorneal epithelial cells which are also to be considered asnon-excitable cells (Watsky et al., 1991). Na+ channels ofthis type are usually inactive at voltage ranges lower than�60mV. Rae and Watsky revealed that there may be asmall steady-state window current at this voltage rangeand these channels could serve as a low-conductance Na+

entry step. They suggested that TTX could influence theelectrophysiological properties of the (bovine) corneal

uit of a whole-cell configuration: Rf: feedback resistor, Vcom: command

ent. The circuit of the amplifier represents a so-called current-to-voltage

breaking through the membrane, the whole-cell configuration is obtained.

te opening and could subsequently increase the serial resistance RS.

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+ 50mV

+ 30mV

– 30mV

– 50mV

– 70mV 3pA

20mS

Fig. 6. Single channel recordings at several voltages from a K+ channel in

an on-cell patch of the apical membrane of rabbit endothelium. [From

Rae and Watsky, Am. J. Physiol. Cell Physiol. 1991; reprinted by

permission from The American Physiological Societyr 1996.]

50

0

-50

Curr

ent (p

A)

-100

-150

0 5

Time (mS)

10

Fig. 5. Whole-cell current traces from a freshly isolated rabbit corneal

endothelial cell containing a voltage-sensitive Na+ current. [From Rae

and Watsky, Am. J. Physiol. Cell Physiol. 1991; reprinted by permission

from The American Physiological Societyr 1996.]


endothelium by which the stroma of the cornea remainsrelatively dehydrated (Rae and Watsky, 1996). Further-more, Fischbarg et al. studied the relative contributions ofcellular mechanisms of Na+ transport and the homeostasisof intracellular [Na+] in cultured bovine corneal endothe-lial cells. They also found an apical voltage-dependentepithelial Na+ channel in these cells and concluded that theNa+ pump flux is associated with these apical Na+

channels (Kuang et al., 2004). However, it is not clearwhether this channel is identical to the TTX-sensitive Na+

channel described by Watsky et al. because no patch-clampdata exist. Therefore, the physiological role of voltage-dependent Na+ channels in the corneal endothelium is stilluncertain.

3.1.2. Potassium channels

Potassium (K+)-selective ion channels are widely dis-tributed and essential in all cell types. Among otherfunctions, K+ channels are predominantly involved insetting the resting membrane potential. In addition, theygenerate electrical signals in excitable cells, and regulatecell volume and cell movement. Importantly, they also playa significant role in K+ secretion (Hebert et al., 2005).There are diverse types of K+ channels such as voltage-gated K+ channels (Kv1.1–Kv1.6) (e.g. delayed rectifiers,A-type voltage-gated K+ channels), inward rectifier K+

channels (Kir1.1–Kir3.1) (preferentially pass K+ ionsinward; steep voltage dependence) (Kubo et al., 2005),and ligand-gated K+ channels such as ATP-dependent K+

channels (KATP) or Ca2+-dependent K+ channels (KCa1)(Chandy and Gutman, 1993; Goldstein et al., 1998). AllK+ channels have dissimilar functions (Desir, 1992;Giebisch, 2001; Faber and Sah, 2003; MacKinnon, 2003;Korn and Trapani, 2005; Hebert et al., 2005). In contrastto sodium channels in human corneal endothelial cells,more is known about K+ channels. Rae and Watsky

described two major K+ currents using patch-clamptechniques (Fig. 6, Rae and Watsky, 1996).K+ currents similar to the family of A-currents are

registered in excitable cells. The A-current is transientafter a depolarising voltage step and is blocked by both4-aminopyridine and quinidine. These currents may play arole in the steady-state activity of the endothelium and itsfluid regulatory function in the cornea. Finally, the authorssuggested that these two currents could be responsible forsetting the �50 to �60mV resting membrane potentialreported for these cells (Rae and Watsky, 1996). Furtherstudies by Rae report the occurrence of a shaw-type

potassium channel (Kv3.3) producing A-currents and aninwardly rectifying potassium channel in corneal endothe-lial cells (Rae and Shepard, 2000a, b). They suggest thatKv3.3 is at least one molecular agent responsible forA-currents in the lens epithelium and corneal endothelium.However, the limitation of these studies is that most patch-clamp measurements were not performed on (human)corneal endothelial cells. Rae et al. investigated Chinesehamster ovary (CHO) cells following transfection withcDNA encoding full-length Kv3.3 (Rae and Shepard,2000a, b). Instead of the corneal endothelium, recentstudies by Lu demonstrated an important function of K+

channels in the corneal epithelium (Lu, 2006). In parti-cular, he elucidated the role of Kv3.4. In conclusion, apotential role for K+ channels in corneal endothelium ispartly still hypothetical. In addition, it is not yet knownwhether investigations performed under in vitro conditionsduring patch-clamp recordings reflect the in vivo situation.

3.1.3. Chloride channels

Chloride (Cl�) channels including ligand-activatedchloride-selective ion channels at synapses (the GABAand glycine-activated channels), as well as voltage-gatedCl� channels are found in various cell types. Theyencompass the so-called ClC family of voltage-dependent

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M

A

B

C

1500

1000

500

200

1500

1000

500

200

1500

1000

500

200

1 2 3 4 5 6 7 8 9

M 1 2 3 4 5 6 7 8 9

M 1 2 3 4 5 6 7 8 9

Fig. 7. Gel electrophoresis of RT-PCR products for members of the ClC

family in the cornea. A: Corneal epithelial cell sample. B: Stromal sample.

C: Corneal endothelial cell sample. Lane M: Molecular weight marker.

Lane 1: ClC-1. Lane 2: ClC-2. Lane 3: ClC-3. Lane 4: ClC-5. Lane 5: ClC-

6. Lane 6: ClC-7. Lane 7: ClC-Ka. Lane 8: NBC positive control. Lane 9:

NBC negative control. [From Davies et al., Mol. Vis. 2004; reprinted by

permission from Molecular Visionr 2004.]


Cl� channels, the cAMP-activated transmembrane con-ductance regulator (CFTR), Ca2+-activated Cl� channels(CaCC), and volume-regulated anion channels (VRAC)(Jentsch et al., 2002; Nilius and Droogmans, 2003; Pusch,2004; Hartzell et al., 2005). Cl� channels play a central rolein fluid movement e.g. in the corneal epithelium, trachealepithelium, and other cells (Rae and Watsky, 1996).However, there is a lack of (patch-clamp) studies of Cl�

channels in corneal endothelial cells. On the other hand,there is evidence from molecular biological studies that Cl�

channels can be expected in the human corneal endothe-lium since maintenance of stromal hydration relies onactive transendothelial anion transport, with bicarbonateand chloride being the major anions carrying the current(Davies et al., 2004). Using reverse transcription polymer-ase chain reaction (RT-PCR), Davies et al. first identifiedthe gene expression profile of members of the ClC family ofchloride channels in freshly isolated samples of rabbitcorneal endothelium, stroma, and epithelium (Fig. 7).Specifically, they detected two Cl� channels, the cysticfibrosis transmembrane conductance regulator (CFTR)and the calcium-activated Cl� channel-1 (CLCA1) andconclude that these channels are likely to be important forthe maintenance of corneal transparency. On the otherhand, it is likely that Cl� channels are also involved inelectrotactic cell migration (Zhao et al., 2006). In summary,there are only a few studies of this channel. The activationmechanisms are conjectural and it is suggested that specifictypes of Cl� channel are operative under physiologicalconditions.

3.1.4. Calcium channels

VOCCs are membrane channels that are specific forcalcium. It is a voltage-dependent cell membrane glyco-proteins selectively permeable to calcium ions. Exocytosisof hormones or other secretory products are triggered byVOCC-dependent rises in the cytosolic free Ca2+ concen-tration (Koizumi and Inoue, 1998; Zeng et al., 1998;Maechler and Wollheim, 1999; Zanner et al., 2002).VOCCs are expressed in various tissues such as skeletalmuscle, cardiac muscle, endocrine cells, neurons, dendrites,and nerve terminals (McDonald et al., 1994; Catterall,2000; Catterall et al., 2005b). Pharmacological andelectrophysiological properties of VOCCs are determinedby the pore-forming a1 subunits. Ten members of theVOCC family are known, which differ in their unique a1subunits (Cav-subunits) (Catterall, 2000; Catterall et al.,2005b). These members are divided into L-type channels(Cav1.1–1.4; dihydropyridine sensitive), P/Q-type channels(Cav2.1, o-Agatoxin sensitive), N-type channels (Cav2.2,o–Conotoxin GVIA sensitive), R-type channels (Cav2.3,former blocker resistant channel, SNX-482 sensitive),and T-type channels (Cav3.1–3.3, low-voltage activated)(Catterall, 2000; Catterall et al., 2005b) (Table 1).

In human corneal endothelial cells, dihydropyridine-sensitive L-type channels have been recently detected bypatch-clamp techniques and calcium measurements. These

studies revealed that these channels are also sensitive toepidermal growth factor (EGF) and endothelin-1 (ET-1)(Hong et al., 2003; Mergler et al., 2003, 2005).Regarding Ca2+ inflow, it is of interest to characterise

the channel types affecting this process since their functionis related to human cornea endothelial proliferation andsurvival. Growth factors such as FGF and EGF areimportant because of their protective effect in cornealepithelial and endothelial cells (Rieck et al., 1995, 2003)and non-corneal cells (Heck et al., 1992; Musallam et al.,2004). Mergler et al. provide evidence that L-type Ca2+

channels in human corneal endothelial cells are partiallyactive near the resting membrane potential, which was

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Table 1

Physiological function and pharmacology of calcium channels

Channel Current Localisation Specific antagonists Cellular functions

Cav1.1 L Skeletal muscle; transverse tubules Dihydropyridines;

phenylalkylamines;

benzothiazepines

Excitation–contraction coupling

Cav1.2 L Cardiac myocytes; smooth muscle

myocytes; endocrine cells; neuronal

cell bodies; proximal dendrites

Dihydropyridines; phenyl

alkylamines; benzothiazepines

Excitation–contraction coupling;

hormone release; regulation of

transcription; synaptic integration

Cav1.3 L Endocrine cells; neuronal cell bodies

and dendrites; cardiac atrial

myocytes and pacemaker cells;

cochlear hair cells



Hormone release; regulation of

transcription; synaptic regulation;

cardiac pacemaking; hearing;

neurotransmitter release from

sensory cells

Cav1.4 L Retinal rod and bipolar cells; spinal

cord; adrenal gland; mast cells



Neurotransmitter release from

photoreceptors

Cav2.1 P/Q Nerve terminals and dendrites;

neuroendocrine cells

o-Agatoxin IVA Neurotransmitter release; dendritic

Ca2+ transients; hormone release

Cav2.2 N Nerve terminals and dendrites;

neuroendocrine cells

o-Conotoxin-GVIA Neurotransmitter release; dendritic

Ca2+ transients; hormone release

Cav2.3 R Neuronal cell bodies and dendrites SNX-482 Repetitive firing; dendritic calcium

transients

Cav3.1 T Neuronal cell bodies and dendrites;

cardiac and smooth muscle

myocytes

None Pacemaking; repetitive firing

Cav3.2 T Neuronal cell bodies and dendrites;

cardiac and smooth muscle

myocytes

None Pacemaking; repetitive firing

Cav3.3 T Neuronal cell bodies and dendrites None Pacemaking; repetitive firing

From Catterall et al., Pharmacol. Rev. 2005b; reprinted with permission from The American Society for Pharmacology and Experimental Therapeuticsr

2005.


assayed by patch-clamp recordings and fluorimetricmeasurements (Fig. 8). Their results suggest that thesecells also express Ca2+ channels potentially lacking avoltage sensor. This corresponds to studies of Ca2+

channels in rabbit corneal epithelial cells (Rich and Rae,1995).

In another study, Rich and Rae observed a non-voltage-dependent Ca2+ permeable channel activity. They couldnot detect whole-cell inward currents carrying Ca2+ orBa2+ (Rich and Rae, 1995). Taken together, humancorneal endothelial cells do not only express VOCCs ofthe L-type.

3.2. Transient receptor potential channels (TRPs)

Generally, store-operated calcium channels (SOCCs) orTRPs represent a diverse group of cation channels that actas cellular sensors of diverse functions. Various differentsubfamilies of TRP channels are known (Montell, 2001;Nilius and Voets, 2005; Montell, 2005; Pedersen et al.,2005; Clapham et al., 2005) (Fig. 9).

All of them are permeable to monovalent cations and themajority to Ca2+. For example, TRP channels include amelastatin-related transient receptor potential subfamily(TRPM), represented by TRPM1–TRPM8 (Harteneck,2005; Kraft and Harteneck, 2005). TRPM8 (originallynamed Trp-p8) is a cold- and menthol-sensing Ca2+-

permeable channel which plays a crucial role in thermo-sensation (McKemy et al., 2002; Peier et al., 2002; Voetset al., 2004; Andersson et al., 2004; Chuang et al., 2004;Brauchi et al., 2004; Weil et al., 2005; Harteneck, 2005;Thebault et al., 2005). There are strong indications thatTRP channels are also involved in many diseases. Forexample, mutations in TRPs are responsible for variouskidney diseases and various cancers (Nilius et al., 2005;Schonherr, 2005).

3.2.1. Calcium-permeable TRPs

Recent studies revealed that there are probably alsotemperature-dependent TRPs expressed in human cornealendothelium (Mergler et al., 2005). Since TRP channels areintimately linked with intracellular Ca2+ signalling, theymay play a significant role in the control of cell cycleprogression, cell migration, and programmed cell death(Clapham et al., 2005). This could also refer to humancorneal endothelial cells. Mergler et al. found by usingfluorescence cell imaging that icilin, an agonist for thetemperature-sensitive TRPM8 had complex dose-depen-dent effects on intracellular Ca2+ concentration [Ca2+]i inhuman corneal endothelial cells (Fig. 10, Mergler et al.,2005). Icilin artificially induced higher cold effects to cellscompared to menthol. There are several reports ontemperature-dependent effects on corneal viability duringstorage (Lindstrom, 1990; Hsu et al., 1999). In this context,

ARTICLE IN PRESS

control Bay K 8644

(5 µM)

100 sec

[Ca2+]i50 nM

recovery

C

0

50

100

150

200

intr

acellu

lar

calc

ium

(nM

)

control

recovery

n = 8

n = 8

n = 7

**

Bay K 8644 (5 µM)

current density (pA/pF)

pipette potential (mV)

-80 -60 -40 -20 20 40

control

Bay K 8644

(5 µM)

Bay K 8644 (5 µM)

control

-70 mV+20 mV

1 pA/pF

15 msec

-2.5

-2.0

-1.5

-1.0

-0.5

A

B

Fig. 8. Identification of L-type Ca2+ channel activity in human corneal

endothelial cells (HCEC-SV40. (A) Effect of extracellular application of

the L-type channel opener BayK8644 (5mM) summarised in a normalised

current/voltage relation (pA/pF versus mV) and as normalised current

traces (pA/pF) (insert) measured in the perforated-patch configuration.

Ba2+ (10mM) was used as charge carrier under extra- and intracellular

Na+- and K+-free conditions. The cells were depolarised from �70 to

+20mV in 10mV steps of 25ms duration. For current/voltage relation,

maximal peak current amplitudes (pA) were normalised by the according

cell capacity (pF) and plotted against the potential of electrical stimulation

(mV). BayK8644 (quadrangles) significantly increased current amplitudes.

(B) Effect of BayK8644 (5mM) on intracellular free Ca2+ concentration

([Ca2+]i) in unstimulated cells. [From Mergler et al., Exp. Eye Res. 2003;

reprinted with permission from Elsevier Ltd.r 2003.]


it could be possible that the activity of putative tempera-ture-sensitive TRP channels such as TRPM8 channels maybe increased at lower storage temperatures. This couldresult in graded shifts of voltage-dependent activationcurves of such TRP channel (Voets et al., 2004).

Interestingly, Rae and Watsky described an outwardlyrectifying K+-selective current assayed by patch-clamptechniques, which could be activated by elevated tempera-tures (Rae and Watsky, 1996). The kind of ion channelsthey detected was not specified. Such an interrelationshipbetween temperature variations may compound the sensi-tivity of the corneal endothelium to damage induced byvarious causes. Importantly, temperature control duringstorage may play an essential role for retaining viability ofstored human corneas. However, further studies arenecessary to confirm this hypothesis. Besides TRPM8expression in human corneal endothelial cells, Mergleret al. suggested that there may be also other TRPsexpressed in human corneal endothelium because H2O2

induces significant rises in [Ca2+]i (Mergler et al., 2005).This Ca2+ rise could be due to activation of TRPM2 assuggested in the literature (Zhang et al., 2003; Kraft et al.,2004). Therefore, human corneal endothelial cells mayexpress a number of TRPs, but the question arises whatpurpose these channels could have under physiologicalconditions in human corneal endothelium. ConcerningTRPM8 in human corneal endothelial cells, it is suggestedthat there may exist an activation or regulation mechanismof TRPM8 other than by temperature. Another possibleregulatory mechanism could be that TRPM8 activity isactivated by G protein-coupled receptors. For example,Kraft and Harteneck described in non-corneal cells thatboth activation of a G protein-coupled receptor and anerve growth factor receptor inhibited menthol- and cold-induced TRPM8 activity (Kraft and Harteneck, 2005).Taken together, the investigation of occurrence andfunction of TRP channels in human corneal endothelialcells is still in the beginning. Initial studies suggested thatthe above-mentioned temperature-sensitive TRPM8 chan-nel is expressed and associated with calcium homeostasis inhuman corneal endothelial cells. In contrast to the cornealendothelium, recent studies by Yang et al. confirmed animportant function of Ca2+-permeable TRPs in thecorneal epithelium. Specifically, they elucidated severalfunctions of TRPC4 in context with growth factors (Yanget al., 2005; Zhang et al., 2006). Generally, it is known thatTRP channels are involved in various fundamental cellfunctions. The determination of their impact in the(patho)physiology of the human corneal endothelium aswell as corneal diseases will be a challenge in biomedicalsciences.

4. Pathophysiology

Altered function of the corneal endothelium is char-acteristic for various corneal diseases such as primarycorneal endotheliopathies (Table 2) (Fig. 11). Thesealterations could also be due to endothelial damagesduring surgical procedures, such as cataract extraction orcorneal transplantation (Bourne, 2003).In general, there is an alteration in function as

distinguished from structural defects. Corneal endothelial

ARTICLE IN PRESS

Fig. 9. Phylogenetic tree of the TRP superfamily. [From Pedersen et al., Cell Calcium 2005; reprinted by permission from Elsevier Ltd.r 2005.]

[Ca2+]i

A

B

100 n

M

100 sec

contr.

control

250

200

150

intr

acellu

lar

calc

ium

(nM

)

100

50

0

washout

washout

**n = 6

n = 6 n = 6

icilin

(10 µM)

icilin (10 µM)

Fig. 10. Effect of the cooling compound icilin on cytosolic free Ca2+ in

human corneal endothelial cells (HCEC-SV40). (A) Extracellular applica-

tion of icilin (10mM) did reversibly increase cytosolic free Ca2+. (B)

Summary of the experiments with icilin (all n ¼ 6). [From Mergler et al.,

Exp. Eye Res. 2005; reprinted by permission from Elsevier Ltd.r 2005.]


cells can be biophysically investigated with the help ofpatch-clamp techniques concerning their electrophysiolo-gical characteristics. So far, a failed ion channel function incorneal endothelial cells leading to an isolated and specifiedcorneal disease or a patho-physiological alteration ofcorneal cells has not been demonstrated. Thus, a detailedinvestigation of ion channel activity in the above-men-tioned corneal diseases and during corneal organ storage isa challenge to better characterise the pathophysiology ofthe corneal endothelium.

4.1. Pump-leak mechanism

The corneal endothelium maintains the stroma of thecornea relatively dehydrated by functioning as both abarrier to fluid movement into the cornea and an activepump that moves ions to draw water osmotically from thestroma into the aqueous humour. The pump-leak mechan-ism corresponds to a combined leaky barrier and fluidpump, and can be measured clinically by fluorophotometryand pachometry. The barrier function can be estimatedfrom the endothelial permeability to fluorescein (Carlsonet al., 1988). The endothelial pump rate can be calculatedfrom the deswelling rate and the endothelial permeability(Bourne et al., 1999). In early Fuchs’ dystrophy, theendothelial pump adapts to the compromised endothelialbarrier, and normal or nearly normal corneal hydration ismaintained. In advanced Fuchs’ dystrophy, however, thepump function of the corneal endothelium decreasesfollowed by a decrease in barrier function (Bourne, 2003)

ARTICLE IN PRESS

Table 2

Primary corneal endotheliopathies

Primary corneal

endotheliopathies

Mechanism discussed References

Fuchs’ dystrophy The pump function of the

endothelium decreases first

and is followed by a decrease

in barrier function. Mutation

in the gene for collagen VIII.

Excessive apoptosis may be an

important mechanism in the

pathogenesis of Fuchs’

dystrophy.

Biswas et al.

(2001); Li et al.

(2001)

Posterior

polymorphous

dystrophy (PPD)

Endothelium contains

epithelial-like cells. Abnormal

cells may appear in isolated

areas, across entire

endothelium. Prognosis of

surgical procedures to improve

vision and lower intraocular

pressure is directly related to

the pathologic endothelium.

Krachmer (1985)

Congenital hereditary

endothelial dystrophy

(CHED)

Disorder of the corneal

endothelium characterised by

bilateral, diffuse corneal

clouding. Pathological and

genetical overlap between PPD

and CHED. Disturbance of

the role of type VIII collagen.

Callaghan et al.,

(1999); Biswas et

al. (2001)

Iridocorneal

endothelial syndrome

(ICE)

Comprised of three syndromes

that are recognised to be

varied manifestations of the

same primary endothelial

disease: essential iris atrophy,

Chandler’s syndrome, and

Cogan–Reese iris nevus

syndrome. The endothelium

has a beaten metal or

cobblestone appearance and

the cells have many

characteristics of epithelial

cells such as micovilli and

cytokeratin markers (Fig. 11).

Share similar genetic

abnormalities with other

primary corneal

endotheliopathies.

Levy et al. (1995);

Anderson (2001)

Intermediate forms Primary endothelial

abnormalities that do not fit

the first four well-defined

entities.

Bourne (2003)

Fig. 11. Example of abnormal endothelial cells in a primary corneal

endotheliopathy such as the ICE syndrome. Note that the ‘ICE cells’ are

enlarged and appear as the ‘negative image’ of normal endothelial cells;

the cell junctions appear light and the cell bodies appear dark (scale

bar ¼ 100mm). [Reprinted by permission from Macmillan Publishers Ltd.:

Eye 2003 (17), 912–918, Nature Publishing Groupr 2003.]


and a subsequent loss of its transparency. Similarly, injuryby ocular surgery could also cause this process.

Fresh isolation of corneas and their dissection is adamaging process resulting in a quick cornea swelling andloss of transparency. At present, patch-clamp technologyhas not been used to characterise this process. Firstinvestigations of the corneal endothelium from a pigshowed alterations in current patterns of putative non-selective cation channels during this injury process(Mergler et al., 2005, unpublished observation) (Fig. 12).

Thus, increases in ion channel activity or alterations ofintercellular junctions are involved in the process of cornealswelling.

4.2. Cell loss

Endothelial cells in the human cornea decline constantlythroughout life (Li, 1985). The human endothelial celldensity is about 6000 cells/mm2 during the first month oflife, but decreases already to about 3500 cells/mm2 by ageof 5 years (Bourne, 2003). Despite a constant loss of cells,normal thickness and transparency are maintained. Addi-tional stress imposed on the endothelium increases with ageand accelerate endothelial cell loss. Corneal endothelial cellloss is particularly accelerated after penetrating kerato-plasty performed patients with Fuchs’ dystrophy (11.2%,annually) and bullous keratopathy (19.3%, annually)(Langenbucher et al., 2002). The mechanisms leading tothis cell loss are not sufficiently characterised so far. Inaddition, there is a still existing ‘‘basic pathology’’. Somestudies described apoptotic-induced events which may beinvolved (Cho et al., 1999; Rieck et al., 2003; Sagoo et al.,2004). The reasons for triggering the apoptotic programmein the human corneal endothelium are not well understood,but metabolic changes in the medium, mechanical stress,endotoxins, the loss of survival factors, and nutrientdeprivation may be involved (Rieck et al., 2003). Inaddition, it has been described that inflammatory cytokinescan induce apoptosis of corneal endothelium (Sagoo et al.,2004). Many intracellular proteins are involved either inthe induction or prevention of apoptosis. For example, thefamily of Bcl-2-related proteins contains important mem-bers of apoptosis-regulatory proteins that act upstream ofthe caspase cascade. They show different activation- andtissue-dependent expression patterns (e.g. lens fibre cells)

ARTICLE IN PRESS

A B

C D

70 mV

2 min

50 min

500 msec

10 n

A

1000 msec

50 min

2 min

16

14

12

10

curr

ent (n

A)

voltage (mV)

8

6

4

2

0

-2

-4-150 -100 -50 0 50 100

10 n

A

- 60 mV

- 120 mV

Fig. 12. Non-selective cation channel current patterns in freshly isolated corneal endothelial cells. The current patterns altered during the patch-clamp

recordings (cornea swelling). (A) Stimulation protocol. (B) Currents before and after cornea swelling (2 and 50min). (C) Current–voltage plot before and

after cornea swelling. (D) Typical light micrograph of the corneal endothelium of a pig before cornea swelling (patch-pipette on the right).


(Dahm, 1999). In particular, Bcl-xL, a member of the Bcl-2-family is able to regulate the membrane permeability andthe release of cytochrome c from mitochondria (Klucket al., 1997; Yang et al., 1997). This protein also confersresistance to a wide variety of pro-apoptotic stimuli. Bag-1,another member of the family of anti-apoptotic genes,exerts its protective effects mainly by binding and stabilis-ing Bcl-2 (Takayama et al., 1995, 1999).

4.3. Are ion channels involved in apoptosis?

Apoptosis may result from a variety of molecularpathways (Fink and Cookson, 2005). In this connection,free radicals, calcium, and oxidative stress play importantroles (Cho et al., 1999; Mergler et al., 2005). Onecharacteristic property of apoptosis is that it is associatedwith increased intracellular calcium levels (Shinomiya etal., 1997; Chami et al., 2001) as well as with various(changed or mostly increased) ion channel activities (seeTable 3). Importantly, permanent slight increases incalcium levels may change regulation of VOCCs in cornealendothelial cells. Experiments have shown the same effectin human retinal pigment epithelial cells (Mergler et al.,1998). Studies of other cell types (e.g. T-lymphocytes,thymocytes) demonstrated that calcium and potassiumchannels are involved in apoptosis (Nagy et al., 1995;

Chvatchko et al., 1996). In addition, it has been identifiedthat an increase in Ca2+ is as an important component ofthe apoptotic pathway from early signalling steps to theactivation of enzymes involved in the execution phaseof apoptosis. The increase is due to the release of Ca2+

from intracellular stores (mitochondria and endoplasmicreticulum) as well as an influx of extracellular Ca2+ ions(Dahm, 1999). Therefore, homeostasis of calcium ions(Ca2+) linking with Ca2+-permeable ion channel activityplays a crucial role in apoptosis. A recent study byLu additionally demonstrated that stress-induced cornealepithelial apoptosis is mediated by K+ channel activation(Lu, 2006). Specifically, UV irradiation induced K+

channel hyperactivity and cell shrinkage (K+ efflux).As described before, there are also clues to putativeactivity of Ca2+-permeable TRP channels such asTRPM2 and TRPM8 in human corneal endothelial cells(Mergler et al., 2005). TRP channel expression could alsobe confirmed in human corneal epithelial cells (Yang et al.,2005). TRPM2 selectively activates by H2O2, whereasTRPM8 currents evoke by cooling agents such asmenthol or icilin. Generally, TRP channel activationresulted in an increase in [Ca2+]i (McKemy et al., 2002;Zhang et al., 2003; Kraft and Harteneck, 2005). Specifi-cally, Zhang et al. assayed by reverse transcriptase-PCRand confocal microscopy of non-corneal cells that a short

ARTICLE IN PRESS

Table 3

Ion channels and apoptosis

Type of ion channel Cell type and roles References

K+ channel Importance of K+ channel activity in T-lymphocyte

proliferation.

Nagy et al. (1995)

ATP-gated ion channel Cells of lymph nodes and thymus. ATP receptor P2X1 may be

selectively involved in the process of immature thymocyte

apoptosis, but not in that of peripheral T lymphocytes.

Chvatchko et al. (1996)

SOC (ICRAC) Calcium influx in non-excitable cells regulates apoptosis. (Parekh and Penner, 1997)

Cav1.3 Retinal pigment epithelial cells (RPE). Changed regulation of

L-type Ca2+ channels lead to retinal degeneration RPE cells

(RCS rat).

(Mergler et al., 1998)

Cl� channel Disordered or altered cell volume regulation by Cl� channels is

associated with apoptosis.

Okada and Maeno (2001)

Voltage- and Ca2+-sensitive

K+ (maxi-K) channel

Activation of K+ channels induces apoptosis in vascular

smooth muscle cells.

Krick et al. (2001)

TRPM2 TRPM2-S is an important physiologic isoform of TRPM2 and

modulates channel activity and induction of cell death by

oxidative stress through TRPM2-L. TRPM2-S inhibited

susceptibility to cell death and onset of apoptosis induced by

H2O2 in cultivated cell lines and human BFU-E-derived cells—

Jurkat cells.

Zhang et al. (2003)

Voltage-gated K+ channel Activation of a delayed rectifier K+ current in differentiating

stem cells is related to apoptosis.

Hribar et al. (2004)

TRPM8 Cultivated prostate cancer cells. Important new ER Ca2+

release channel, potentially involved in a number of Ca2+ and

store-dependent processes in prostate cancer epithelial cells,

including those that are important for prostate carcinogenesis,

such as proliferation and apoptosis.

Skryma et al. (2000); Thebault et al. (2005)

Cav1.3, Ca2+-permeable

TRPs?

Cultured corneal endothelial cells express L-type channels.

Cyto-protective molecules such as Bcl-xL or HO-1 reduced

intracellular Ca2+ levels by inhibition of Ca2+-permeable

channel activity and are very effective inhibitors of apoptosis.

Mergler et al. (2003) and an unpublished

observation by Ritter et al. (2006)

Voltage-dependent anion

channel (VDACpl)

VDACpl of neuronal hippocampal cell line (HT22) cells was

activated during apoptosis; channel block prevented apoptosis.

Akanda and Elinder (2006)

K+ channel (Kv3.4) Stress-induced corneal epithelial apoptosis mediated by K+

channel activity.

Lu (2006)


isoform of TRPM2 (TRPM2-S) has an important physio-logical function and modulates channel activity andinduction of cell death by oxidative stress through the fulllength of isoform of TRPM2 (TRPM2-L) (Zhang et al.,2003). Pharmacological blockade of TRPM8 in non-corneal cells, or suppression of its expression by smallinterfering RNA targeted to TRPM8, results in apoptosis(Zhang and Barritt, 2004). Before, Komuro et al. havedemonstrated that the TUNEL assay identifies cellsundergoing apoptosis in corneas stored at 4 1C (Komuroet al., 1999). Cell death occurs by both apoptosis andnecrosis in stored corneas, with apoptosis appearing topredominate. Therefore, inhibition of apoptosis mayincrease cell survival and thereby prolong the maximumperiod of storage of corneas before transplantation(Komuro et al., 1999). However, a possible involvementof temperature-sensitive receptors such as TRPM8 or othertemperature-sensitive TRPs such as TRPV1 in theseconditions is still unknown. As mentioned before, calciumhomeostasis may play an important role in corneal tissuesas well as intracellular pH (Grant and Acosta, 1996).Calcium is a regulator of various cellular functions and it

has a crucial function as second messenger for many signal(space) transduction pathways (Putney, Jr. and Bird, 1993;Clapham, 1995). Thereby, intracellular enzymes and ionchannel conductance of the cell membranes are regulatedby intracellular calcium concentration in many cell typesincluding corneal endothelial cells (Strauss et al., 1997; Wuet al., 1997; Mergler et al., 1998; Munaron and Fiorio,2000; Mergler and Strauss, 2002; Mergler et al., 2003). Inconclusion, there is strong evidence that Ca2+-permeableion channels are expressed in human corneal endothelialcells and these channels are very likely to be associated withapoptotic events.

5. Clinical relevance

Corneal endothelial dysfunction is most frequentlycorrelated with an accelerated loss of endothelial cells.A number of factors can contribute to the endothelial cellloss, including aging, trauma, ocular surgery, Fuchs’endothelial dystrophy, and Type I diabetes.This accelerated cell loss may eventually result in

compromised corneal function and decompensation that


requires intervention. Currently, the only treatment avail-able for treatment of the endothelial cell loss is cornealtransplantation (keratoplasty). Other possible forms oftreatment that are under investigation include arresting thecell loss, transplanting corneal endothelial cells, and/orstimulating fluid secretion of the remaining endothelialcells. In all of these instances, a better characterisation andimproved knowledge on ion channels may have an impacton clinical management. Some recent aspects are brieflysummarised in respect to corneal endothelial electrophy-siology and ion channels.

5.1. Endothelial cell preservation during corneal banking

The focus on improving corneal culturing prior totransplantation is to minimise corneal endothelial cell lossduring corneal organ culture. Prior to keratoplastypreservation of the donor endothelium during storage iscrucial. Already during donor tissue storage, an irreversibleendothelial cell loss of 10–30% occurs. Subsequently, up to40% of all organ-cultivated donor corneas are unsuitablefor transplantation (Directory of the European Eye BankAssociation, EEBA). The present lack of donor corneasfurther aggravates the problem. Several mechanisms havebeen suggested as responsible for this finding. For example,the role of apoptosis may be an important point. Aspreviously mentioned (Section 4.3), one characteristicproperty of apoptosis is that it is associated with increasedintracellular calcium levels, as well as with changes of(mostly increased) ion channel activities. Therefore, athorough study of ion channel characteristics undervarious conditions may help to better understand theirinfluence on apoptotic mechanisms during corneal en-dothelial cell loss.

In this respect, growth factors are important. Examplesinclude EGF, platelet-derived growth factor (PDGF), andfibroblast growth factor (FGF). Various effects of thesefactors on human corneal endothelial cells have beencharacterised, for example, modulation of cell proliferation(Bednarz et al., 1996a, b). Importantly, growth factors areassociated with various ion channel activities in cornealcells (Yang et al., 2003; Mergler et al., 2003, 2005; Yanget al., 2005; Zhang et al., 2006).

Currently, corneal grafts are either preserved usinghypothermic storage (4 1C, Optisol) predominantly in theUS, or organ culture at 34 1C, which is preferred in Europe.In this context, a number of studies focused on growthfactors supplemented by storage media. Growth factorshave been added to DexSol corneal storage medium(Chiron Ophthalmics, Irvine, California). A multi-centretrial showed that the use of corneas stored in DexSol withadded human EGF and insulin in corneal transplantationresulted in a better clinical outcome (concerning endothe-lial cell loss). This is comparable with that observed inpatients receiving DexSol-stored corneas. However, therewere no clinically and statistically significant differences inpostoperative endothelial morphometric parameters (Lass

et al., 1994). A further corneal storage medium isdesignated as Optisol (Chiron Ophthalmics, Irvine, Cali-fornia) and contains dextran, chondroitin sulphate, vita-mins, and precursors of adenosine triphosphate (adenosine,inosine, and adenine). It helps to preserve corneal integrityfor up to two weeks at 4 1C, thereby permitting flexibility inthe use of donor tissue for corneal transplantation. Inaddition, Optisol storage yields thinner tissue compared toprevious methods of storage which allow for more accurateevaluation and faster recovery following surgical manip-ulation (Lindstrom et al., 1992).In general, it is proven that hypothermic preservation of

the cornea alters the corneal endothelium and affects itsintegrity more as compared to organ culture at highertemperature. An understanding of the mechanisms respon-sible for the sensitivity is of interest to optimise the limitednumber of available donor materials. Recent studies ofhuman corneal endothelial cells revealed that temperature-sensitive channels such as the cold receptor TRPM8 areputatively expressed in these cells because a selectiveagonist of this channel induced significant calcium re-sponses (Mergler et al., 2005). Moreover, a TRP channel ofanother subtype family was detected (TRPC4) (Yang et al.,2005). This channel activity is linked with EGF. Finally,previous studies demonstrated that distinct growth factors(e.g. EGF, bFGF) protect corneal endothelial cells (Schil-ling-Schon et al., 2000; Rieck et al., 2003; Mergler et al.,2005). Specifically, a recent electrophysiological study oncultivated human corneal endothelial cells revealed thatEGF overcomes oxidative stress-induced increases in[Ca2+]i by H2O2 (Mergler et al., 2005) (Fig. 13). Thus,the presence of EGF may improve cornea preservation andthereby permit to use more corneas.On the other hand, the bovine spongiform encephalo-

pathy (BSE) crisis has abandoned the use of bovine-derivedserum for human corneal banking. In order to prevent thedanger with contamination and the risk of BSE, putativeimprovements of organ culture omitting foetal calf serumwere investigated (serum-free organ culture) (Camposam-piero et al., 2003). However, in vitro studies showed thatendothelium in serum-free media will have a shortened lifespan but also altered non-uniform histological appearance(Bednarz et al., 2001; Hempel et al., 2001; Moller-Pedersenet al., 2001a, b). Therefore, development of novel techni-ques to circumvent the requirement for serum are animportant challenge. Currently, there is a lack of electro-physiological studies elucidating the function of ionchannels in the storage conditions. Therefore, investigationof ion channel characteristics might be an importantcontribution to better characterise the alterations duringserum-free culture since these storage conditions maymodify the physiology of corneal endothelial cells.

5.2. Corneal endothelial cell transplantation

New approaches to transplanting human as well asanimal (rabbit, rat) corneal endothelial cells have been

ARTICLE IN PRESS

A

B

Fig. 13. EGF overcomes oxidative stress-induced rises in [Ca2+]i by H2O2

in cultivated human corneal endothelial cells. (A) Extracellular application

of EGF (10 ng/m1) and H2O2 (1mM) slightly increased [Ca2+]i. After

washout of EGF, H2O2-induced Ca2+ increase was not influenced.

Following washout of H2O2 intracellular free Ca2+ declined. (B)

Summary of the experiments with H2O2 and EGF. [From Mergler et al.,

Exp. Eye Res. 2005; reprinted by permission from Elsevier Ltd.r 2005.]


developed by several investigators (Bohnke et al., 1999;Engelmann et al., 1999; Amano, 2003; Ishino et al., 2004;Mimura et al., 2004a, b). For example, transplantation ofadult human or porcine corneal endothelial cells ontohuman recipients in vitro has been investigated. Inaddition, a method for grafting endothelial cells isolatedfrom organ-cultured adult human corneas onto thedenuded Descemet’s membrane of human recipients hasalso been developed (Bohnke et al., 1999; Engelmann et al.,1999). Engelmann et al. (1999) demonstrated that trans-plantation of immortalised human corneal endothelial cellsonto the Descemet’s membrane of recipient corneasestablishes a new endothelial cell layer. This had not onlythe morphological characteristics of a physiological one,but also and most importantly possessed its functionalcapacity to regulate stromal hydration (Aboalchamat et al.,1999). Other studies demonstrated that cultured humancorneal endothelial cells transplanted from an adult humandonor cornea by means of a collagen sheet could retaintheir function of corneal dehydration in a rabbit model.This suggests the feasibility of transplantation for cornealendothelial cell dysfunction using cultured human cornealendothelial cells with a collagen sheet (Mimura et al.,

2004b). Finally, more recent studies have demonstratednovel strategies to culture and proliferate human cornealendothelial cells and fabricate endothelial sheets ex vivo.

These sheets are morphologically and functionally similarto the native corneal endothelium (bioengineered humancorneal endothelium for transplantation) (Lai et al., 2006;Hsiue et al., 2006; Sumide et al., 2006). However, tissue-engineering strategies are not easy to perform because thecellular components either cannot survive or they lose theirfunctional phenotype rapidly within a biomaterial matrixupon implantation or in culture. In this context, tempera-ture-responsive culture dishes are helpful (Sumide et al.,2006). This point may be of interest regarding temperature-sensitive TRP channels in human corneal endothelial cells.Taken together, the knowledge of bioengineered humancorneal endothelial cell sheets is increasing and this shouldalso become available for clinical application in the future.

5.3. Optimising of rinse solutions for intraocular surgery

Generally, rinse solutions are used at all intraocularoperations. They play an important role in maintainingphysiological conditions during surgery. Its composition ofsalts, temperature, and its pH value are parameters thatinfluence the viability of corneal endothelial cells. In thiscontext, a recent study investigated a specific washing(ophthalmic) solution for extra- and intraocular operations(containing (mmol/L) NaCl, 109.5; KCl, 10; CaCl2, 3.25;MgCl2, 1.5; Na-acetate, 47.5; Na-citrate, 6.587; pHadjusted to 7.3) in order to find whether changes inintracellular free Ca2+ are dependent on sodium andpotassium concentrations (Mergler et al., 2005). Artificialoxidative stress on cultivated human corneal endothelialcells was induced by exposure to H2O2 and measuredintracellular calcium concentration [Ca2+]i. Low levels ofH2O2 (non-toxic; 0.1mM) induced reversible non-deleter-ious [Ca2+]i transients, whereas higher concentrations from10mM increased [Ca2+]i to considerably higher Ca2+

levels, which compromised cell viability (Mergler et al.,2005). All measurements were performed in a sodium- andpotassium-free solution and were identical to thoseobtained in the above-mentioned ophthalmic solution.This indicates that the H2O2-induced increase of intracel-lular calcium is independent of sodium and potassiumconcentration. Therefore, non-selective cation channelscould be involved in causing an intracellular calcium influxirrespective of the presence or absence of these ions. Takentogether, further electrophysiological investigations ofspecific rinse solutions on the physiology could help toidentify which optimal rinse solutions are most appropriatefor use in intraocular operations.

6. Summary, conclusions, and future directions

Investigation of the electrophysiology and ion channelexpression of the corneal endothelium by highly sensitivemeasuring methods is a new field that may open new


perspectives. It not only will help to better understandphysiological functions of the cornea, but also may havedirect clinical implications. Interestingly, some earlierstudies of a putative Na+ channel do exist, but have notbeen subsequently followed (Watsky et al., 1991). Inaddition, further studies elucidate the importance ofexploring the function of definite ion channels, inparticular, investigations of K+ and Ca2+ channels inhuman corneal endothelial cells (Rae et al., 1989, 1990;Watsky et al., 1992; Green et al., 1994; Rae and Watsky,1996; Mergler et al., 2003). Recently, investigationsindicate the presence of putative and temperature-sensitiveTRP channels in human corneal endothelial cells (Mergleret al., 2005). However, a possible role of these TRPsregarding the storage temperature of corneas is stillunknown. Another important fact is that apoptosis isassociated with changed ion channel activity and intracel-lular calcium levels. In this context, free radicals andoxidative stress play important roles. Some studies of non-corneal and corneal cells have demonstrated that growthfactors may overcome oxidative stress-induced rises inintracellular calcium by oxidants. For clinical purposes, thebasic electrophysiological characteristics of the humancorneal endothelium and its relationship to the cornealphysiology may help to improve the keratoplasty outcomeas well as corneal culturing prior to transplantation. It alsomay facilitate approaches to transplant human cornealendothelial cells. Furthermore, rinse solutions for intrao-cular surgery can be optimised after detailed investigationsof its effects on ion channels in human corneal endothelialcells. Whether potential malfunctions of ion channelsare relevant to specific corneal diseases or cell loss stillhas to be elucidated in future studies. In conclusion, amajor challenge will be to clarify the impact of electro-physiology and ion channels in context with an improvedcorneal endothelial cell viability in vivo. This is a processwhich should be facilitated by the use of newly developedion channel modulators, growth factors and transgenicanimals.

Acknowledgements

The authors appreciate the helpful comments by PeterReinach (Ph.D.), State University of New York, College ofOptometry, 33 West 42nd Street, New York, NY 10036,USA and Vinodh Kakkassery (MD), Schepens EyeResearch Institute, Harvard Medical School, 20 StanifordSt., Boston, MA 02114, USA.

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