Experimental Eye Research 116 (2013) 337e349

Contents lists available at ScienceDirect

Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer

Functional significance of thermosensitive transient receptor potential melastatin channel 8 (TRPM8) expression in immortalized human corneal endothelial cellsq Stefan Mergler a, *, Charlotte Mertens a, Monika Valtink b, Peter S. Reinach c, Violeta Castelo Székely d, Nefeli Slavi c, Fabian Garreis e, Suzette Abdelmessih f, Ersal Türker a, Gabriele Fels a, Uwe Pleyer a a

Charité e Universitätsmedizin Berlin, Campus Virchow-Clinic, Department of Ophthalmology, Augustenburger Platz 1, 13353 Berlin, Germany Institute of Anatomy, Faculty of Medicine Carl Gustav Carus, TU Dresden, Fetscherstr. 74, 01307 Dresden, Germany c Department of Pharmacology, University of Sao Paulo, School of Medicine, Ribeirao Preto, Brazil d Charité e Universitätsmedizin Berlin, International Graduate Program Medical Neurosciences, Charitéplatz 1, 10117 Berlin, Germany e Department of Anatomy II, University of Erlangen-Nürnberg, Universitätsstraße 19, Erlangen, Germany f Charité e Universitätsmedizin Berlin, Campus Virchow-Clinic, Department of Gastroenterology, Augustenburger Platz 1, 13353 Berlin, Germany b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 February 2013 Accepted in revised form 3 October 2013 Available online 14 October 2013

Human corneal endothelial cells (HCEC) maintain appropriate tissue hydration and transparency by eliciting net ion transport coupled to fluid egress from the stroma into the anterior chamber. Such activity offsets tissue swelling caused by stromal imbibition of fluid. As corneal endothelial (HCE) transport function is modulated by temperature changes, we probed for thermosensitive transient receptor potential melastatin 8 (TRPM8) functional activity in immortalized human corneal endothelial cells (HCEC12) and freshly isolated human corneal endothelial cells (HCEC) as a control. This channel is either activated upon lowering to 28  C or by menthol, eucalyptol and icilin. RT-PCR and quantitative real-time PCR (qPCR) verified TRPM8 gene expression. Ca2þ transients induced by either menthol (500 mmol/l), eucalyptol (3 mmol/l), or icilin (2e60 mmol/l) were identified using cell fluorescence imaging. The TRP channel blocker lanthanum III chloride (La3þ, 100 mmol/l) as well as the TRPM8 blockers BCTC (10 mmol/l) and capsazepine (CPZ, 10 mmol/l) suppressed icilin-induced Ca2þ increases. In and outward currents induced by application of menthol (500 mmol/l) or icilin (50 mmol/l) were detected using the planar patch-clamp technique. A thermal transition from room temperature to z 18  C led to Ca2þ increases that were inhibited by a TRPM8 blocker BCTC (10 mmol/l). Other thermosensitive TRP pathways whose heterogeneous Ca2þ response patterns are suggestive of other Ca2þ handling pathways were also detected upon strong cooling (z10  C). Taken together, functional TRPM8 expression in HCEC-12 and freshly dissociated HCEC suggests that HCE function can adapt to thermal variations through activation of this channel subtype. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: human corneal endothelium transient receptor potential melastatin 8 channel intracellular Ca2þ planar patch-clamp technique

1. Introduction The human corneal endothelium (HCE) prevents the cornea from becoming translucent by offsetting the natural tendency of

Abbreviations: HCEC-12, immortalized human corneal endothelial cell line; TRPM, transient receptor potential melastatin; TRPA, transient receptor potential ankyrin; BCTC, N-(4-tert.butyl-phenyl)-4-(3-chloropyridin-2-yl) tetrahydropyrazine-1(2H)-carboxamide; CPZ, capsazepine; CAP, capsaicin. q Contract grant sponsor: Berliner Sonnenfeld-Stiftung, Contract grant number: 89745052 Contract grant sponsor: Allergan, Contract grant number: 20081006. * Corresponding author. Tel.: þ49 (0)30 450 559648; fax: þ49 (0)30 450 559948. E-mail addresses: [email protected], [email protected] (S. Mergler). 0014-4835/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.exer.2013.10.003

the stroma to imbibe fluid and swell. Such protection is rendered by its mediation of outward fluid flow coupled to net ion transport activity from the stroma into the anterior chamber. Their regulation is in part mediated by different receptors whose activation induces Ca2þ signaling (Bonanno, 2012). These functions are temperature sensitive but the expression of some subtypes of thermosensitive transient receptor potential (TRP) channels suggests that the HCE cells are buffered to some extent from functional declines by a compensatory change in the regulation of Ca2þ homeostasis. This suggestion is based on our characterization of several members in the vanilloid subfamily of TRP channels (i.e TRPV1-3) in a human corneal endothelial cell line (HCEC-12) that are activated by a rise in temperature above 42  C (Mergler et al., 2010). We hypothesized

338

S. Mergler et al. / Experimental Eye Research 116 (2013) 337e349

that HCE cells may be also poised to withstand temperature lowering since it was shown that preservation of intact corneal thinness is better maintained at 23  C than 31  C due to improved preservation of barrier function (Sandboe et al., 2003). Accordingly, we probed here for functional TRP melastatin 8 (TRPM8) expression since this channel becomes activated by temperature lowering from those in the physiological range. The melastatin TRP subfamily M is another member of the seven thermosensitive TRP subfamilies. This subfamily comprises eight subtypes (TRPM1-8) (Fonfria et al., 2006). TRPM8 channels in corneal nerve terminals play a role in maintaining ocular surface health through control of tear fluid secretion (Parra et al., 2010). This Ca2þ permeable channel is also termed “menthol-receptor” and its activity is modulated by exposure to moderate cold and can be activated by cooling agents such as menthol, icilin or eucalyptol (Bautista et al., 2007; Chuang et al., 2004; Peier et al., 2002a; Voets et al., 2004, 2007; McKemy et al., 2002; Behrendt et al., 2004). While TRPM8 can be activated by moderate cooling (temperature threshold z 28  C), another subfamily constituent is TRPA1, which has a temperature threshold of z17  C. TRPA1 was proposed to be a detector of noxious cold in nociceptive afferents (McKemy, 2005). The cooling compound icilin is a known TRPM8 and TRPA1 agonist (McKemy et al., 2002). However, it is less potent in activating TRPA1 than TRPM8 (Story et al., 2003). Functional TRP expression patterns in various eye tissues including corneal epithelium and endothelium were recently reviewed (Pan et al., 2011). Here, we describe TRPM8 gene expression in HCEC-12 and normal human corneal endothelial cells (HCEC) as a control. Its functional activity was characterized based on changes in ionic currents and Ca2þ transients induced by thermal variations known to activate this channel. Its presence suggests that in vivo corneal endothelial function may adapt to some thermal stresses through activation of this pathway. 2. Materials and methods 2.1. Materials BCTC and fura-2AM were purchased from TOCRIS Bioscience (Bristol, United Kingdom). Capsazepine and icilin were purchased from Cayman Chemical Company (Ann Arbor, Michigan, U.S.A.). Medium and supplements for cell culture were purchased from Life Technologies Invitrogen (Karlsruhe, Germany) or Biochrom AG (Berlin, Germany). Accutase was purchased from PAA Laboratories (Pasching, Austria). All other reagents were purchased from Sigma (Deisenhofen, Germany). 2.2. Culture of human corneal endothelial cells The human corneal endothelial cell line HCEC-12 was immortalized by transfection with pRNS, a plasmid carrying the SV40 small t- and large T-antigen (Bednarz et al., 2000, 1995). Cells were obtained from DSMZ, Braunschweig, Germany. Normal HCEC were isolated from a donor cornea that was rendered unsuitable for transplantation (Engelmann et al., 1988). HCEC-12 as well as normal HCEC were grown in Medium 199/Ham’s F12 supplemented with 5% FCS, 20 mg/ml insulin, 20 mg/ml ascorbic acid, 10 ng/ ml human recombinant FGF-2 and 100 IU/ml penicillin/streptomycin. Cells were grown in a humidified atmosphere containing 5% CO2 at 37  C. They were subcultured using accutase, whose activity was quenched with serum-supplemented growth medium. Cells were centrifuged at 100  g and plated at a split ratio of 1:10 (HCEC-12) or 1:4 (normal HCEC) onto T25 flasks. Medium was changed three times per week.

2.3. Polymerase chain reaction Total RNA was isolated from human corneal endothelial cell populations HCEC-12, HCEC-B4G12, HCEC-H9C1 and normal HCEC up to passage P3, as well as from the human positive control cell populations LNCaP (prostate carcinoma), 647V (bladder carcinoma), and BON-1 (neuroendocrine tumor) using the RNA II kit (Machereye Nagel, Oensingen, Switzerland) or the RNeasyÒ Mini Kit (Qiagen, Hilden, Germany) according to the supplier’s instructions. For RTPCR, 2 mg RNA were digested with DNAse I (Fermentas, St. LeonRot, Germany) and subjected to reverse transcription using the RevertAid First strand cDNA synthesis kit (Fermentas) according to the manufacturer’s instructions. PCR was carried out with 2 ml cDNA mixture in a 25 ml total volume using the Taq DNA Polymerase kit (InvitrogenÔ). Each reaction containing 2.5 ml 10 reaction buffer, 1 ml 50 mM MgCl2, 0.25 ml 10 mM dNTPs, 0.125 ml Taq DNA polymerase and 0.25 ml 10 pmol forward and reverse primer. Primers used were 50 -CCTGTTCCTCTTTGCGGTGTGGAT-30 and 50 0 TCCTCTGAGGTGTCGTTGGCTTT-3 , yielding a 621 bp product. Nontemplate PCR control samples were set up with RNAse- and DNAse-free water instead of cDNA. PCR was initiated with 1  95  C for 5 min, followed by 35 cycles of 95  C for 15 s, 65  C for 30 s, 72  C for 30 s, and finalized with 1  72  C for 7 min followed by a temperature hold at 4  C in a GeneAmp PCR System 9700 thermocycler (Applied Biosystems, by Life Technologies, Darmstadt, Germany). As template control, b-actin was determined using primers 50 GATCCTCACCGAGCGCGGCTACA-30 and 50 -GCGGATGTCCACGTCACACTTCA-30 , yielding a 298 bp product. Samples were electrophoresed on a 1.2% agarose gel at 80 V for 30 min and visualized using ethidium bromide under UV light. The 621 bp bands were cut out, extracted from the agarose using the Jetquick Gel Extraction Spin kit from Genomed (Loehne, Germany) and sequenced (Eurofins MWG Operon, Ebersberg, Germany). For qPCR, RNA was reverse transcribed into cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Darmstandt, Germany). The expression pattern of TRPM8 gene was validated by qPCR using the Bio-RAD CFX96Ô Real-time system (Bio-Rad, Munich, Germany) and TaqManÒ Gene Expression Assays (Applied biosystems) for TRPM8 (#Hs00375481_m1) and GAPDH (#4326317E-1105041), with Gene expression master mix (FastStart TaqManÒ probe master; Roche Applied Biosystems, Mannheim, Germany), according to the supplier’s instructions. The experiment was performed in a 96 well plate and initialized at 95  C for 10 min followed by 45 cycles of 95  C for 15 s and 62  C for 30 s. For each sample, triplicate reactions were performed with 30 ng cDNA per reaction in a final volume of 10 ml. The housekeeping gene GAPDH was used as an internal control. Mean CT values were calculated. For the relative quantification (RQ), the “relative” expression values were calculated from the delta delta CT values using the formula: 2DDCT. This was normalized to the housekeeping gene GAPDH as an internal control and to the reference sample. The reference sample was set to the sample with the highest CT value, i.e. with the lowest mRNA concentration/content. The experiment was performed twice and the data were analyzed in Excel (Microsoft Deutschland GmbH, Unterschleibheim, Germany) and GraphPad Prism version 5 for Windows (GraphPad Software, San Diego California USA). 2.4. Intracellular calcium imaging For calcium imaging experiments, cells were pre-incubated with culture medium containing 1 mmol/l fura-2AM for 20e40 min at 37  C. Fura-2/AM reaction was quenched with a pre-warmed (37  C) Ringer-like (control) solution containing 150 mmol/l NaCl, 6 mmol/l CsCl, 1 mmol/l MgCl2, 10 mmol/l glucose, 10 mmol/l HEPES and 1.5 mmol/l CaCl2 (pH 7.4). Coverslips with fura-2 loaded

S. Mergler et al. / Experimental Eye Research 116 (2013) 337e349

cells were thoroughly rinsed in this solution to remove any cell debris or dead cells. Washed coverslips were mounted in a chamber containing Ringer-like solution on the stage of an inverted microscope (Olympus BW50WI, Olympus Europa Holding GmbH, Hamburg, Germany) and left for a few minutes to adapt to room temperature (z 22  C). A digital photometry imaging system (T.I.L.L. Photonics, Munich, Germany) at 340 nm and 380 nm excitation wavelengths was used for fluorescence recording at 510 nm emission wavelength. The measuring field contained a cluster of fura-2-loaded cells as shown in Fig. 1. The fluorescence ratio (f340/ f380) provides an index of intracellular Ca2þ concentration ([Ca2þ]i) (Grynkiewicz et al., 1985). Results are expressed in terms of f340/ f380 nm  SEM with n values indicating the number of experiments per data point. The measurements lasted between 8 and 20 min depending on the experimental design [Ca2þ]i baseline level measurements were obtained during the first 3e4 min, followed by isosmotic replacement with Ca2þ-free Ringer-like solution (1 mmol/l EGTA) which led to declines in [Ca2þ]i. Three min later, 1.5 mmol/l Ca2þ was added to increase [Ca2þ]i (data not shown). The reversibility of Ca2þ changes evaluated cell viability since restoration of baseline f340/f380 nm is indicative of functional

A

B

Fig. 1. TRPM8 gene and functional expression in HCEC. (A) RT-PCR revealed gene expression of TRPM8 in different HCEC populations. M: 100 bp DNA ladder, lane 1: normal cultured HCEC, lane 5: HCEC-B4G12, lane 6: HCEC-H9C1, lane 7: HCEC-12. As negative controls, no reverse transcriptase was used during cDNA synthesis (lane 2 for normal HCEC, lane 8 for HCEC lines), and PCR was performed without template (lane 4). Human prostate carcinoma cells (LNCaP) served as positive controls (lane 3, lane 9). In accordance with DNA marker, the distinct DNA bands are visible at 621 bp for TRPM8 and at 298 for b-actin. (B) Quantitative PCR results for TRPM8 in HCEC-12 cells and the TRPM8-expressing control cell line BON-1 (human neuroendocrine tumor cell line) were normalized to the cell line 647V (human bladder carcinoma cell line) (RQ ¼ 1). Messenger RNA quantity was determined from CT values. The higher the value the less mRNA is present.

339

intracellular Ca2þ regulation (Mergler et al., 2012b). Dimethyl sulfoxide (DMSO) vehicle effects were not encountered provided its concentration did not exceed 0.1%. 2.5. Planar patch-clamp recording Whole-cell currents were measured using the planar patchclamp technique (port-a-patchÓ) (Nanion, Munich, Germany) (Bruggemann et al., 2008; Milligan et al., 2009). The intracellular solution contained 50 mmol/l CsCl, 10 mmol/l NaCl, 2 mmol/l MgCl2, 60 mmol/l CsF, 20 mmol/l EGTA and 10 mmol/l HEPES, pH adjusted to 7.2 with KOH (osmolarity: 288 mOsmol). Csþ in the solution blocks potassium channel outward currents, and the hydrate-covering of fluoride can block possible anion chloride channels. The extracellular solution contained 140 mmol/l NaCl, 4 mmol/l KCl, 1 mmol/l MgCl2, 2 mmol/l CaCl2, 5 mmol/l D-glucose monohydrate and 10 mmol/l HEPES, pH adjusted to 7.4 with NaOH (osmolarity: 298 mOsmol). A cell suspension was prepared using accutase, and cells were further dissociated by gentle trituration with a pipette. Cells were gently pipetted onto a microchip on the setup that was prepared with the measuring solutions and had a resistance of 2e5 MU, which is comparable to a patch-pipette resistance. Suction was applied automatically by a software controlled pump in order to move a single cell onto the aperture of the microchip. Further suction pulses led to a break in the cell membrane to achieve a whole-cell configuration. Membrane currents were recorded using an EPC 10 amplifier with Patchmaster software version 2.5 for Windows (HEKA, Lambrecht, Germany). Membrane capacitance and access resistance of HCEC-12 were calculated with Patchmaster software. A mean access resistance of 29  19 MU (n ¼ 32) and a mean cell membrane capacitance of 12  1 pF (n ¼ 32) was observed in the whole-cell configuration. Series resistance errors as well as fast and slow capacity transients were compensated by the software controlled patch-clamp amplifier. More specifically, the series resistance was compensated (z 70e90%) after breaking into whole-cell configuration. Only cells with leak currents below 200 pA were used and current recordings were leak-subtracted. In some cases, leak currents were over-compensated by the software, which led to positive currents at 60 mV that were not retained for evaluation. In most cases, reducing the software-imposed opposing conductivity by 1e2 nS lessened overcompensated leak currents. Liquid junction potentials were always compensated for prior to experimentation. All experiments were performed at room temperature (z22  C) in an airconditioned room. When drugs were applied from DMSOcontaining stock solutions, DMSO was kept < 0.1% to avoid interfering with patch-clamp recordings. The holding potential was set to 0 mV to eliminate any possible contributions by voltagedependent Ca2þ or Naþ channel activity. Whole-cell currents were recorded for 400 ms using voltage steps ranging between 60 and þ130 mV (10 mV increments). The resulting currents were normalized by dividing the current (pA) amplitude by cell membrane capacitance (pF) to obtain current density ([pA/pF]). Voltage changes without steps (voltage ramps) were also used, and in some cases, time courses of currents were recorded using the ramp protocol every 5 s. All plots were generated with SigmaPlot software version 12.3 (Systat Software, San Jose, California, U.S.A.) and an electrophysiology module (Systat, Bruxton). Bar charts were plotted with GraphPad Prism version 5. 2.6. Statistics For normally distributed values according to the Gaussian distribution, statistical analysis with Student’s t-test was performed (parametric paired or unpaired two-sample t-test). The parametric-

340

S. Mergler et al. / Experimental Eye Research 116 (2013) 337e349

Fig. 2. Cold receptor mediation of cooling agent-induced Ca2þ entry into HCEC-12. Ca2þ was measured in HCEC-12 following fura-2-loading and [Ca2þ]i monitoring using fluorescence cell imaging in a Ringer-like solution. The reagents were added at the time points indicated by arrows. Data are means  SEM of 4e24 experiments. Ca2þ baselines were recorded as controls (n ¼ 10 to n ¼ 30). (A) and (B) Fura-2 loaded human corneal endothelial cells (HCEC-12) at low cell density (A) and high cell density (B) viewed using fluorescence emission at 510 nm. Scale bar is 100 mm. Typically, a small cluster of healthy appearing cells was selected from the cover slip for calcium imaging by adjusting the aperture. Healthy cells were chosen on a basis of smooth membranes, even and bright fura-2 loading, and shape. Typically, cells with projections are the most well anchored and therefore best for imaging. (C) ()-Menthol (500 mmol/l, filled circles) selectively activated TRPM8 induced Ca2þ influxes (n ¼ 8). No Ca2þ changes were observed without application of ()-menthol (open circles, control; n ¼ 12). (D) Eucalyptol (3 mmol/l, filled circles) induced Ca2þ influxes (n ¼ 5). No Ca2þ changes were observed without application of eucalyptol (control; n ¼ 10). (E) Summary with ()-menthol and eucalyptol (500 mmol/l and 3 mmol/l) in HCEC-12. The asterisks (*) indicate significant differences (at the

S. Mergler et al. / Experimental Eye Research 116 (2013) 337e349

A

B

1.26

341

C

1.26

1.26

n = 18

1.22 n = 19 1.20

control HCEC-12 (≈ 22 °C)

1.24

20 μM icilin

1.22 n = 11 1.20

10 μM CPZ

1.18

1.18 0

200

400

0

600

200

400

1.24 20 μM icilin 1.22 n = 18 1.20

10 μM BCTC

1.18 600

0

200

time (sec)

time (sec)

D

HCEC-12 (≈ 22 °C) fluorescence ratio f 340 /f 380

20 μM icilin

1.24

fluorescence ratio f 340 /f 380

fluorescence ratio f 340 /f 380

HCEC-12 (≈ 22 °C)

control (120 sec)

E

F

1.22

(18)

***

(18) ## ###

1.22 (18)

(11) (11)(11)

1.20

### ###

(18) (18) (18)

1.18 r cke CPZ BCTC t blo 10 μM u o 0 μM h w it with with 1

60 μM icilin

icilin (600 sec)

n=7

1.21

n=6 1.20 control normal HCEC (> 28 °C)

1.19

1.22 fluorescence ratio f340/f380

**** 1.24

icilin (400 sec)

fluorescence ratio f340/f380

fluorescence ratio f340/f380

1.26

600

control (200 sec)

20 μM icilin (400 sec) 20 μM icilin (600 sec)

400

time (sec)

* **

1.21

(7)

(7) (7) 1.20

1.19

0

200

400

600

time (sec)

Fig. 3. Icilin induced Ca2þ entry blocked by TRPM8 channel blockers. Reagents were added at the time points indicated by arrows. Data are means  SEM of 7e18 experiments. Ca2þ baselines were recorded as controls (n ¼ 6e19). (A) Icilin (20 mmol/l, filled circles) selectively activated TRPM8/TRPA1 induced Ca2þ influxes (n ¼ 18) in HCEC-12. No Ca2þ changes were observed without application of icilin (control; n ¼ 19). (B) With the mixed TRPM8/TRPV1 channel blocker CPZ (10 mmol/l), the icilin-induced Ca2þ increase fell (n ¼ 11). (C) With the TRPM8 channel blocker BCTC (10 mmol/l), the icilin-induced Ca2þ increase was also clearly suppressed (n ¼ 18). (D) Summary of the experiments with 20 mmol/l icilin. Asterisks (*) indicate significant differences (at the minimum p < 0.01; paired Student’s t-test) between control (Ca2þ base level at 120 s) and icilin (at 400 s and 600 s). The hashes (#) indicate significant differences (at the minimum p < 0.01; unpaired Student’s t-test) between icilin-induced Ca2þ increases with and without the TRPM8 channel blockers. (E) In normal HCEC, icilin (60 mmol/l, filled circles) also induced Ca2þ influxes under nearly physiological conditions (>28  C; n ¼ 7) confirming functional cold receptor expression in HCEC. No Ca2þ changes were observed without application of icilin (control; n ¼ 6). (F) Summary of the experiments with 60 mmol/l icilin. Asterisks (*) indicate significant differences (at the minimum p < 0.05; paired Student’s t-test) between control (Ca2þ base level at 200 s) and icilin (at 400 s and 600 s).

free Wilcoxon-Mann-Whitney rank-sum test was used for assessing whether one of two samples of independent measurements tended to have larger values than the other. The number of replicates is indicated in each case in brackets. In case of too different variances, Welch’s correction was used. P values < 0.05 of all tests were considered as significant. 3. Results 3.1. TRPM8 mRNA expression We used LNCaP (prostate tumor cell line) as a positive control to verify TRPM8 expression in HCEC-12 (Valero et al., 2011), its

clonal daughter cell lines HCEC-B4G12 and HCEC-H9C1, and normal HCEC by RT-PCR. The results shown in Fig. 1A confirm that TRPM8 gene expression is evident in HCEC cell lines and normal HCEC. Further assurance of its identity is based on sequence identity of the isolated PCR product and GenBank data. In order to quantify TRPM8 gene expression in HCEC-12, qPCR was performed with HCEC-12 and BON-1 (neuroendocrine tumor cell line) as a positive control (Fig. 1B) (Mergler et al., 2007). As reference cell line, 647V (human bladder carcinoma cell line) was used, as it had the lowest mRNA content (RQ ¼ 1). As compared to the reference cell line, TRPM8 expression was greater in HCEC-12 (RQ ¼ 2.235  1.153) and BON-1 (RQ ¼ 5.085  2.044) than in the reference cell line.

minimum p < 0.05; unpaired Student’s t-test) between control (Ca2þ base level at 300 s) and menthol/eucalyptol (at 300 s). (F) Icilin (60 mmol/l, filled circles) selectively activated TRPM8 induced Ca2þ influxes (n ¼ 9). No Ca2þ changes were observed without application of icilin (control; n ¼ 30). (G) Icilin-induced dose dependent increases in [Ca2þ]i. 2e 60 mmol/l clearly induced Ca2þ increases, whereas 200 nmol/l had only minor effects (n ¼ 4 to n ¼ 24). (H) In the presence of the TRP channel blocker La3þ (100 mmol/l), the icilininduced Ca2þ increase was suppressed (n ¼ 5). (I) Summary of the experiments with 60 mmol/l icilin in HCEC-12. The asterisks (*) indicate significant differences (at the minimum p < 0.05; paired Student’s t-test) between control (Ca2þ base level at 200 s) and icilin (at 400 s). The hashes (#) indicate significant differences (p < 0.005; unpaired Student’s t-test) between icilin-induced Ca2þ increases with and without 100 mmol/l La3þ.

342

S. Mergler et al. / Experimental Eye Research 116 (2013) 337e349 80

B

500 μM (-)-menthol

current density (pA/pF)

current density (pA/pF)

A

80

130 mV

60

2 mM Ca

C TRPM8

40

20

0

Ca -free -60 mV -20

-60 0

50

100

150

200

60 40 20 0 -20

20

60

100 140 V (mV)

-20

time (sec)

D

E

control 500 μM (-)-menthol recovery

F

control 500 μM (-)-menthol recovery

G current density (pA/pF)

1.2 1.0 0.8 I (nA)

0.6 0.4 0.2 0.0 -0.2 -60

-40

-20

0

20

40

60

80

100

120

120 100 80 60 40 20 0 -20

(12)

(12)

**

140

600 400 200 0 -200 -400

rol

ol

t con

50

M 0μ

m

h ent

0m

ry

r

ve eco

3 +1

130 mV

current amplitude (% of control)

current amplitude (% of control)

I

-600

V

V

m -60

-60 mV

(7)

(7)

(12)

V (mV)

H

**

(12)

400

200

0

-200

trol

ol

nth

con

M 0μ

me

ry

ove

rec

50

Fig. 4. Menthol-activated nonselective cation channel currents. (A) Experimental design (whole-cell configuration of the planar-patch clamp technique). The holding potential (HP) was set to 0 mV to avoid any voltage-dependent ion channel currents. (B) Time course of whole-cell currents at 60 mV (lower trace) and 130 mV (upper trace) showing the current activation by 500 mmol/l () menthol in HCEC-12. The currents were normalized to capacitance to obtain current density (pA/pF) (C) Original traces of menthol activated TRPM8 channel responses to voltage ramps from 60 mV up to þ130 mV (500 ms, with leak current subtraction) in the whole-cell configuration of the planar patch-clamp technique. Currents are shown before application (labeled as A) and during application of 500 mmol/l ()-menthol (labeled as B and C). The currentevoltage relations were obtained from time

S. Mergler et al. / Experimental Eye Research 116 (2013) 337e349

3.2. TRPM8 functional activity Intracellular calcium levels were evaluated based on the ratio of fluorescence intensities (f340/f380) in fura2-loaded HCEC-12 (Fig. 2A, B). Menthol, a specific TRPM8 agonist (Kim et al., 2009; Bandell et al., 2006) increased this ratio to 1.205  0.002 (n ¼ 8) (300 s) (Fig. 2C) whereas in another group of controls it remained constant at 1.200  0.001 after the same period (n ¼ 12; p < 0.05). Similar results were obtained with eucalyptol, (Fig. 2D) another TRPM8 agonist, which has an EC50 value of 7.7 mmol/l (Behrendt et al., 2004). Three mmol/l eucalyptol induced an increase to 1.244  0.019 (n ¼ 5) while its control was invariant at 1.201  0.0003 (n ¼ 10; p < 0.01, Fig. 2D and E). In contrast, the super-cooling agent icilin is both a TRPM8 and TRPA1 agonist (Rawls et al., 2007). In some other tissues, its EC50 values range from ¼ 0.36e66.7 mmol/l (McKemy et al., 2002). We used 0.2e 60 mmol/l icilin (Fig. 2G). With 60 mm/l iclin, the ratio clearly increased up to 1.241  0.009 while its control was 1.201  0.001 (n ¼ 9; p < 0.0001, Fig. 2FeG). On the other hand, preincubation with 100 mmol/l lanthanumeIIIechloride (La3þ), a broad spectrum TRP channel blocker, reduced the icilin-induced rise to 1.216  0.007; (n ¼ 5; p < 0.0001, Fig. 2HeI). Twenty mmol/l icilin increased the ratio from 1.200  0.002 to 1.220  0.014 at 400 s and 22  C (n ¼ 18; p < 0.0001, Fig. 3A). With other TRPM8 blockers, CPZ and BCTC (both at 10 mmol/l), the rise decreased to 1.202  0.003 (n ¼ 11; p < 0.0001; unpaired tested) and 1.205  0.009, respectively (n ¼ 18; p < 0.0001) (Behrendt et al., 2004; Xing et al., 2007) (Fig. 3BeD). With normal cultured HCEC, at a bath temperature above 28  C 60 mM icilin increased the f340nm/f380nm ratio from 1.200  0.0001 to 1.207  0.0021 at 600 s (n ¼ 7; p < 0.05, Fig. 3E) (McKemy et al., 2002). Such a rise is comparable to that obtained with HCEC-12 at room temperature. Taken together, these results are indicative of functional TRPM8 activity in both normal HCEC and immortalized HCEC-12. 3.3. Icilin and menthol activate whole-cell channel currents To identify the underlying currents associated with TRPM8 activation, cells were patched in the whole cell configuration using the planar-patch-clamp technique (Fig. 4A). The holding potential was set to 0 mV to eliminate any possible contributions by voltagedependent Ca2þ and Naþ channel activity (Bruggemann et al., 2006, 2008). At positive pipette potentials, ()-menthol (500 mmol/l) activated large outwardly rectifying currents that were larger than those at negative voltages. Time course measurements along with plots of the corresponding current voltage relationships at the indicated time points A, B and C are shown in Fig. 4B and C. Similar current responses were obtained using a step stimulation voltage protocol (depolarization from 60 mV to þ130 mV in 10 mV steps for 400 ms). The increases in inward current induced by menthol are consistent with the rises in Ca2þ influx in a Ca2þ containing extracellular solution (Fig. 4DeE). Mean current voltage traces generated by the current responses to step voltage changes are shown in Fig. 4F. Fig. 4G summarizes the menthol experiments. Specifically, the inward currents at 60 mV increased from 7  1 pA/pF to 16  3 pA/pF (n ¼ 12; p < 0.001). Recovery at a

343

later time reached 5  1 pA/pF (n ¼ 7; p < 0.05). On the other hand, the outwardly rectifying whole-cell currents increased from 47  8 pA/pF to 95  13 pA/pF (n ¼ 12; p < 0.001) with a recovery to 44  7 pA/pF (n ¼ 7; p < 0.05). Maximal negative current amplitudes induced by a voltage step from 0 mV to 60 mV (% of control) and corresponding maximal positive current amplitudes induced by a voltage step from 0 mV to þ130 mV are shown in Fig. 4I and H. Similar results were obtained with the icilin (50 mmol/l) (Fig. 5). However, the icilin-induced whole-cell currents reached higher levels and were irreversible (Fig. 5AeB). Fig. 5C provides a summary of the icilin results and Fig. 5DeF show the increases in whole cell currents induced by this agonist as a function of the imposed voltage protocol. The mean current voltage relationships show that at 60 mV, the inward currents increased from 47  17 pA/pF to 54  15 pA/pF (n ¼ 15; p < 0.05). At 130 mV, the outwardly rectifying currents increased from 113  33 pA/pF to 194  39 pA/ pF (n ¼ 15; p < 0.001, Fig. 5G). Notably, a less selective TRPM8 channel blocker (CPZ, 10 mmol/l) reduced the icilin-induced outward and inward currents (Fig. 5FeI). Maximal negative current amplitudes were induced by a voltage step from 0 mV to 60 mV (% of control; Fig. 5H). Inward currents decreased from 395  104% of control to 116  35%. Maximal positive current amplitudes were induced by a voltage step from 0 mV to þ130 mV (Fig. 5I). The accompanying outward currents decreased from 334  51% of control to 160  40% (both n ¼ 6; p < 0.05). Overall, these icilin and menthol activated whole-cell currents reflect TRPM8 channel activity. 3.4. Activation of TRPM8 channel by moderate cold To directly delineate thermosensitive TRPM8 activity, the temperature was lowered to z 18  C in the presence and absence of the selective antagonist, 10 mmol/l BCTC (Fig. 6). Whereas moderate cooling below z 25e28  C activates TRPM8, cooling below 17  C activates additionally TRPA1 (Tominaga and Caterina, 2004). Moderate cooling from room temperature (22  C) to z 18  C, which excluded TRPA1 thermo-activation caused the f340nm/f380nm ratio to rise from 1.200  0.002 to 1.210  0.011 within 150 s (n ¼ 10; p < 0.01) (Fig. 6B). Preincubation with 10 mmol/l BCTC, blocked this rise, which is consistent with functional TRPM8 expression (1.200  0.005; n ¼ 6; p < 0.05) (Fig. 6C). Interestingly, the [Ca2þ]i level after cooling to 14  C (Fig. 6A) reached higher levels than that after moderate cooling to z 18  C. This rise is instead suggestive of the involvement of another type of Ca2þ regulatory pathway of unknown identity besides TRPA1 as we could not detect its gene expression. 3.5. Strong cooling induced various Ca2þ responses During exposure to z 10  C, La3þ (500 mmol/l) reduced the ratio from 1.203  0.091 (n ¼ 24) to 1.130  0.030 at 250 s (n ¼ 4; p < 0.01, Fig. 7C). Fig. 7B provides individual traces of the different Ca2þ responses to such a stress. Fig. 7A shows the mean trace and the corresponding temperature curve (n ¼ 24) whereas Fig. 7D summarizes these results. Starting at 20e23  C, the temperature

points indicated by letters A, B and C which are shown in panel (B). The currents were normalized to capacitance to obtain current density (pA/pF). (D) Nonselective cation channel currents induced by depolarization from 60 mV to 130 mV in 10 mV steps (400 ms) after establishing the whole-cell configuration (with leak current subtraction). (E) Increased cation channel currents in the presence of 500 mmol/l ()-menthol. (F) Effects of ()-menthol are summarized in a current/voltage plot (IeV plot, n ¼ 4 to n ¼ 5). All values are reported as means  SEM. For the current/voltage relation, maximal peak current amplitudes were plotted against the voltage (mV). The upper trace (filled circles) was obtained in the presence of 500 mmol/l ()-menthol and the lower trace (open circles) without menthol. A recovery could be observed (filled triangles). (G) Summary of the experiments with ()-menthol. The asterisks (*) indicate statistically significant differences of in- and outward currents with and without ()-menthol (n ¼ 7 to n ¼ 12; p < 0.05; paired tested). (H) Maximal negative current amplitudes induced by a voltage step from 0 mV to 60 mV are depicted in percent of control values before application of 500 mmol/l ()-menthol. (I) Maximal positive current amplitudes induced by a voltage step from 0 mV to þ130 mV are depicted in percent of control values before application of 500 mmol/l ()-menthol.

Fig. 5. Icilin-activated nonselective cation channel currents. (A) Time course of whole-cell currents at 60 mV (lower trace) and 130 mV (upper trace) showing the current activation by 50 mmol/l icilin in HCEC-12. The currents were normalized to capacitance to obtain current density (pA/pF) (B) Original traces of icilin activated TRPM8/TRPA1 channel responses to voltage ramps from 60 mV up to þ130 mV (500 ms, with leak current subtraction). Currents are shown before application (labeled as A) and during application of 50 mmol/l icilin (labeled as B and C). The currentevoltage relations were obtained from time points indicated by letters A, B and C which are shown in panel (A). The currents were normalized to capacitance to obtain current density (pA/pF). (C) Summary with icilin. The asterisks (*) indicate statistically significant differences of in- and outward currents with and without icilin (n ¼ 15; at the minimum p < 0.05; paired tested). (D) Nonselective cation channel currents induced by depolarization from 60 mV to 130 mV in 10 mV steps (400 ms) after establishing the whole-cell configuration (with leak current subtraction). (E) Increased cation channel currents in the presence of 50 mmol/l icilin. (F) Suppressed icilin-induced cation channel currents in the presence of mixed TRPM8/TRPV1 channel blocker CPZ (10 mmol/l). (G) Effects of icilin are summarized in a current/voltage plot (IeV plot, n ¼ 4 to n ¼ 5). All values are reported as means  SEM. For the current/voltage relation, maximal peak current amplitudes were plotted against the voltage (mV). The upper trace (filled circles) was obtained in the presence of 50 mmol/l icilin and the lower trace (open circles) without icilin. 10 mmol/l CPZ suppressed the icilin-induced increase of currents (filled triangles). (H) Maximal negative current amplitudes induced by a voltage step from 0 mV to 60 mV are depicted in percent of control values before application of 50 mmol/L icilin. Icilin-induced inward currents could be clearly suppressed in the presence of 10 mmol/l CPZ. (I) Maximal positive current amplitudes induced by a voltage step from 0 mV to þ130 mV are depicted in percent of control values before application of 50 mmol/l icilin. Icilin-induced outwardly rectifying currents could be clearly suppressed in the presence of 10 mmol/l CPZ.

30 25 20 15 10 5

room temperature n=7

1,28 1,26

cooling (~ 14 °C)

B fluorescence ratio f 340 /f 380

1,30

temperature (°C)

fluorescence ratio f 340 /f 380

A

1,24 n=7 1,22 control 1,20

345

room temperature

1,30

n = 10

1,28 1,26

moderate cooling (~ 18 °C)

1,24

n = 10 1,22 control

1,20

n = 10

n = 10 1,18

0 0

200

400

1,18

600

0 0

200

time (sec)

room temperature

30 25 20 15 10 5

n=6

1,28 1,26

moderate cooling (~ 18 °C)

D

1,24 n=6

1,22 1,20 10 μM BCTC

1,18

control (120 sec) moderate cooling (350 sec) moderate cooling (600 sec)

0

***

1.28

200

400

(7)

*

1.20

(10) (6)

(10)

1.22

TC t BC

ou

with

**

**

1.24

600

time (sec)

#

(7)

1.26

(6) (6)

(10)

(7)

1.18 0

600

1.30

fluorescence ratio f340/f380

1,30

400

time (sec)

temperature (°C)

fluorescence ratio f 340 /f 380

C

30 25 20 15 10 5

temperature (°C)

S. Mergler et al. / Experimental Eye Research 116 (2013) 337e349

)

4°C

(~1

ou

TC t BC

with

)

8°C

(~1

with

M

10 μ

C BCT

)

8°C

(~1

Fig. 6. Ca2þ transients caused by moderate cold exposure. The thermal changes were carried out at the time points indicated by arrows. Data are mean  SEM of 6e10 experiments. (A) Temperature reduction from z22  C to z 14  C resulted in Ca2þ elevation. Traces show intracellular Ca2þ levels in several neighboring cells with moderate cooling (filled circles, n ¼ 10) and without cold (open circles, n ¼ 10). The corresponding temperature course is shown above the Ca2þ traces (n ¼ 10). (B) Temperature lowering from z23  C to z 18  C led to similar results at lower levels. (C) With the TRPM8 channel blocker BCTC (10 mmol/l), the cold-induced Ca2þ increase was suppressed (n ¼ 6). (D) Summary with moderate cold stimulation. The asterisks (*) indicate significant differences (at the minimum p < 0.05; paired Student’s t-test) between control (Ca2þ base level at 120 s) and moderate cold (at 350 and 600 s). The hash (#) indicates a significant difference (p < 0.05; unpaired Student’s t-test) between cold-induced Ca2þ increase with and without 10 mmol/l BCTC.

fell to z 10  C (Fig. 7E). These declines reversed upon warming back to the initial temperature range substantiate TRPM8 involvement. 3.6. Icilin increased capsaicin-induced Ca2þ influx TRPM8 and TRPV1 are coexpressed in HCEC-12 as in various other tissues. We determined if they interact with one another (Mergler et al., 2012a; Crawford et al., 2009; Kobayashi et al., 2005). Cells were pre-incubated with icilin for 30 min. Upon addition of 20 mmol/l capsaicin (CAP) at 400 s, the icilin-induced [Ca2þ]i rise increased further from 1.210  0.003 (n ¼ 9) to 1.234  0.006 (n ¼ 7; p < 0.01, Fig. 8A, summary in Fig. 8B). Fig. 9AeC show whole-cell current responses following the aforementioned step voltage stimulation protocol. CAP (20 mmol/l) increased both outwardly rectifying and inward currents (Fig. 9B). Mean current voltage relationships obtained with the aforementioned voltage step protocol are shown in Fig. 9D along with a summary in Fig. 9E. Specifically, the inward currents at 60 mV rose from 7  1 pA/pF to 18  4 pA/pF (n ¼ 6e7; p < 0.05). Outward currents increased from 30  8 pA/pF to 96  33 pA/pF (n ¼ 6e7; p < 0.01). These increases were normalized to their control values to reduce

variability between individual measurements. Maximal negative current amplitudes were induced by a voltage step from 0 mV to 60 mV (% of control; Fig. 9F). CAP (20 mmol/l) increased inward currents by 210  37% (n ¼ 5; p < 0.05). The corresponding maximal positive current amplitudes induced by a voltage step from 0 mV to þ130 mV revealed, that outward currents increased by 267  45% (n ¼ 5; p < 0.05 (Fig. 9G). Neither inward nor outward currents were altered by replacing 20 mmol/l CAP with 20 mmol/l icilin. Taken together, even though icilin increased CAP-induced Ca2þ entry the whole-cell current did not consistently rise. This disconnect suggests that TRPV1-TRPM8 interaction is not readily demonstrable in HCEC-12 even though it exists in some ocular tumors. 4. Discussion Our results provide convincing evidence that functional TRPM8 expression occurs in the HCE. This conclusion is warranted since along with TRPM8 gene expression we identified Ca2þ transients and underlying currents indicative of its activation by documented agonists and changes in temperature. These

n=4 1.2

1.1 500 μM La

1.0 0

0

cold stimulation (~ 10 °C)

1.3

1.2

1.1 n = 24

0

100 200 300 400 500

fluorescence ratio f 340 /f 380

fluorescence ratio f340/f380

** 1.3

(5) (5) (5)

(4)

(4) (4)

1.1

1.6

1.0

n = 24

n = 24 1.2

1.1

0 0

100 200 300 400 500 time (sec)

25

1.5

15

1.4

10 5

1.3 1.2

n=7

1.1 cold stimulation (< 10 °C) 200

cold stimulation (~ 10 °C)

400

600

800

1000

0

1200

time (sec)

10

50

0

0

μM

20

n=7

0

La

30

μM

La

a tL ou ith

cold

30 25 20 15 10 5

room temperature

1.0

w

1.3

100 200 300 400 500

E

1.4

1.2

room temperature

time (sec)

control (120 sec) cold stimulation (250 sec) cold stimulation (470 sec)

(24) (24)(24)

1.4

1.0

1.0

time (sec)

D

C

temperature (°C)

cold

1.4

temperature (°C)

n=4 1.3

B

fluorescence ratio f 340 /f 380

fluorescence ratio f 340 /f 380

30 25 20 15 10 5

fluorescence ratio f 340 /f 380

room temperature

1.4

temperature (°C)

A

Fig. 7. Complex Ca2þ responses to a strong cold. The thermal changes were carried out at the time points indicated by arrows. Data are mean  SEM of 7e24 experiments. (A) With the TRP channel blocker La3þ (500 mmol/l), no cold-induced positive Ca2þ influxes could be detected (n ¼ 4, z23  C room temperature). (B) Temperature reduction from z22 to 24  C to z 10  C resulted in heterogeneous Ca2þ responses. Single traces show intracellular Ca2þ levels in several neighboring cells following cold stimulation (n ¼ 24). (C) The averaged trace shows intracellular Ca2þ following cold stimulation (filled circles). The corresponding temperature course is shown above the Ca2þ trace (both n ¼ 24). (D) Summary with strong cold stimulation and La3þ (100 and 500 mmol/l). The asterisks (*) indicate a significant difference (p < 0.01; unpaired Student’s t-test) between cold-stimulated Ca2þ change without and with 500 mmol/l La3þ at 250 s. (E) Averaged trace shows intracellular Ca2þ level following repeated cold stimulation from room temperature (z22  C or z 20  C) to z 10  C (filled circles). The corresponding temperature course is shown above the mean Ca2þ trace (both n ¼ 7). Interestingly, at temperatures near 22  C or 20  C, cold exposure only led to a reduction of intracellular Ca2þ concentration.

control (120 sec) without icilin (n = 9) with 20 μM icilin (n = 7)

A

20 μM CAP (400 sec)

B

20 μM CAP (590 sec)

1.28

# 1.28

1.26

20 μM CAP

1.24 1.22 1.20

n=9

1.18

fluorescence ratio f340/f380

fluorescence ratio f 340 /f 380

n=7

***

##

(7)

1.26

** (7)

1.24

*

(9)

(9)

1.22

(11)

(9)

(7)

1.20 1.18

0

100

200

300 time (sec)

400

500

600

ut

ho wit

in

in

icil

M

h

wit

μ 20

icil

Fig. 8. Capsaicin (CAP)-induced Ca2þ influx in HCEC-12 modulated by icilin. Ca2þ was measured in HCEC-12 following fura2-loading and [Ca2þ]i monitoring using fluorescence cell imaging in a Ringer-like solution. 20 mmol/l CAP was added at the time point indicated by an arrow. Data are means  SEM of 7e9 experiments. Experiments were carried out with or without the TRPM8/TRPA1 channel agonist icilin (20 mmol/l). (A) Without icilin, CAP (20 mmol/l, open circles) selectively activated TRPV1 induced Ca2þ influxes (n ¼ 9). In cells pre-incubated in 20 mmol/l icilin, the CAP-induced Ca2þ influxes (filled circles; n ¼ 7) were at higher levels compared to experiment without icilin. (B) Summary of the experiments with 20 mmol/l CAP and 20 mmol/l icilin in HCEC-12. The asterisks (*) indicate significant differences (at the minimum p < 0.05; paired Student’s t-test) between control (Ca2þ base level at 120 s) and CAP (at 400 and 590 s). The hashes (#) indicate significant differences (at the minimum p < 0.05; unpaired Student’s t-test) between CAP-induced Ca2þ increases with and without 20 mmol/l icilin.

S. Mergler et al. / Experimental Eye Research 116 (2013) 337e349

347

Fig. 9. CAP- and icilin-activated nonselective cation channel currents. Currents were induced by depolarization from 60 mV to 130 mV in 10 mV steps (400 ms) after establishing the whole-cell configuration (with leak current subtraction). (B) Increased in- and outward cation channel currents with the TRPV1 agonist CAP (20 mmol/l). (C) Wash out of CAP and subsequent application of the TRPM8/TRPA1 agonist icilin did not change the current patterns. (D) Effects of CAP and icilin are summarized in a current/voltage plot (IeV plot, n ¼ 3 to n ¼ 5). All values are reported as means  SEM. For the current/voltage relation, maximal peak current amplitudes were plotted against the voltage (mV). The upper trace (filled circles) was obtained with 20 mmol/l CAP and the lower trace (open circles) without CAP. 20 mmol/l icilin did not change the CAP-induced current increases (filled triangles). (E) Summary with CAP and icilin. The asterisks (*) indicate significant differences of inward currents with and without CAP or icilin (n ¼ 5 to n ¼ 7; p < 0.05; unpaired tested). The hashes (#) indicate significant differences in outward currents with and without CAP (n ¼ 6; p < 0.01; ManneWhitney test). (F) Maximal negative current amplitudes induced by a voltage step from 0 mV to 60 mV are expressed as percent of control values before application of 20 mmol/l CAP. CAP-induced inward currents were not influenced after washout of CAP and subsequent application of 20 mmol/l icilin. (G) Maximal positive current amplitudes induced by a voltage step from 0 mV to 130 mV are expressed as percent of control values before application of 20 mmol/l CAP. CAP-induced outward currents were not influenced after washout of CAP and subsequent application of 20 mmol/l icilin.

results are relevant to the in-vivo condition since many of the responses elicited by TRPM8 activation in HCEC-12 and its clonal derivatives HCEC-B4G12 and HCEC-H9C1 (Valtink et al., 2008) also occurred in normal HCEC. Besides showing that the electrophysiological properties of freshly isolated primary cultures of HCEC are comparable to those described in HCEC-12, we found that all observed changes in the different endothelial cell types were induced by either icilin or a temperature decrease, and were very similar to each other (Mergler et al., 2003). Furthermore,

TRPM8 activation of Ca2þ transients and whole-cell current responses could be attenuated during exposure to either BCTC or CPZ (Xing et al., 2007; Malkia et al., 2009; Behrendt et al., 2004). Use of the immortalized HCEC lines is relevant, because they mimic more closely a differentiated cell morphology than cultures of normal HCEC. This difference is a consequence of the fact that normal cells have a limited lifespan in culture and undergo dedifferentiation or transitional processes in vitro (Bednarz et al., 2000).

348

S. Mergler et al. / Experimental Eye Research 116 (2013) 337e349

4.1. Menthol receptor activity

4.4. Potential role of TRPM8 in assessing HCE cell viability

Identification of TRPM8 protein expression remains somewhat problematic since antibodies used for this purpose have questionable selectivity (Mahieu et al., 2007), Nevertheless, TRPM8 gene expression is strongly supported by our obtained RT-PCR and qPCR results in both freshly isolated cultured HCEC and different immortalized counterparts (Fig. 1AeC). Furthermore the correspondence in the different cell populations between the changes induced by menthol clearly shows that TRPM8 is functional (Figs. 2 and 4). Menthol-induced Ca2þ influx is a hallmark of TRPM8 expression (Malkia et al., 2009). The effects of menthol are also consistent with those induced by eucalyptol, which is another less potent and efficacious TRPM8 agonist (McKemy et al., 2002; Behrendt et al., 2004). However, the menthol mode of action is complex and still only poorly understood (Peier et al., 2002b; Bautista et al., 2007). This agonist induced outwardly rectifying whole-cell currents and inward currents with a reversal potential of z 0 mV consistent with its behavior as a nonselective cation channel (Voets et al., 2004). The outwardly rectifying current changes correspond to those described in some other tissues (Voets et al., 2004; Weil et al., 2005; McKemy et al., 2002). However, the lower current density levels and outward rectification found in HCEC-12 are probably due to lower TRPM8 expression levels than those in other tissues or TRPM8- expression systems.

Our results showing preservation of TRPM8 functional expression in different immortalized HCEC lines and in freshly isolated cultured HCEC prompts the suggestion that it could be used as a more sensitive marker of endothelial viability than conventional staining and cell counting, which are currently used by eye banks. Cell counting is relatively insensitive since corneal thinning remains rather constant despite large declines in endothelial cell numbers. Screening for previously identified thermosensitive either TRPV1, TRPV3, TRPV4 and now TRPM8 activity may be advantageous since in some locales viable corneas are in limited supply. In summary, there is a close correspondence between TRPM8 gene expression and its functional activity in normal and immortalized HCEC populations. Characterization of its functional activity included identification of agonist-induced Ca2þ transients and underlying currents. Such responses were replicated by temperature changes known to activate TRPM8. Furthermore, preincubation with a selective TRPM8 antagonist blocked these activation responses. Functional TRPM8 expression in HCE may help to explain why previously described endothelial thinning activity in isolated corneas is better maintained at 23  C than at 31  C.

4.2. Icilin increases intracellular Ca2þ through TRPM8 channels Icilin is a more efficacious and potent TRPM8 agonist than menthol (McKemy et al., 2002; Chuang et al., 2004). Icilin irreversibly increased Ca2þ influx and whole-cell currents in both HCEC-12 (Fig. 5AeB) and in normal cultured HCEC under conditions similar to those in vivo (Fig. 3E). These Ca2þ rises correspond to those described in other cell types (Kochukov et al., 2006; Mergler et al., 2007). Their irreversibility suggests that either HCEC are more sensitive to icilin and/or less able than other cell types to reverse the Ca2þrises towards a baseline level (Chuang et al., 2004; Mergler et al., 2007). Nevertheless, TRPM8 involvement was affirmed by showing that preincubation with TRPM8 antagonists, BCTC and CPZ, blocked icilin-induced Ca2þ influx and whole-cell currents in HCEC-12. However, CPZ is a mixed TRPV1 and TRPM8 antagonist (Vriens et al., 2009; Behrendt et al., 2004; Xing et al., 2007) and the effects of CPZ may be complex since there is also functional TRPV1 expression in HCEC-12 (Mergler et al., 2010). In contrast, BCTC is a more selective antagonist. In TRPA1 (/) mice, it suppressed icilin-induced responses by corneal nerve fiber cells (Hirata and Oshinsky, 2012; Parra et al., 2010). Taken together, all of the icilin-induced Ca2þ response patterns in HCEC-12 and normal HCEC described here are comparable to those in previous observations of immortalized HCEC (Mergler et al., 2005) and non-corneal and tumor cell types (Kochukov et al., 2006; Zhang and Barritt, 2004; Mergler et al., 2007). 4.3. Non-physiological cold stimulation At temperatures of z 14  C or 10  C, Ca2þ rises occurred which may involve other plasma membrane delimited thermosensitive receptors (Mergler et al., 2010). However, a role for TRPA1 is open to question since we did not verify TRPA1 gene expression. On the other hand, lowering the temperature below 10  C repeatedly resulted in lowering of intracellular Ca2þ levels. Whether these declines are attributable to stimulation of Ca2þ efflux or an increase in its uptake into a fura2 inaccessible compartment remains to be determined.

Acknowledgments This study was supported by Charité research funds and in part by DFG Pl 150/14-1 as well as by Pharm-Allergan GmbH (Ettlingen, Germany). Stefan Mergler is supported by DFG Me 1706/13-1 and DFG Me 1706/14-1 concerning TRP channel related research projects. Peter Reinach is supported by USP fellowship. The planar patch-clamp set up was supported in part by Berliner SonnenfeldStiftung (Stefan Mergler). The authors thank Kerstin Pehlke (Anatomy, TU Dresden) and Noushafarin Khajavi (Experimental Ophthalmology, Charité Berlin) for technical assistance as well as Carsten Grötzinger (Gastroenterology), Olaf Straub and Norbert Kociok (both Experimental Ophthalmology, Charité Berlin) for helpful discussions. Finally, the authors appreciate very much the support by the fellow students Ekaterina-Maria Lyras and Abhilash Dwarakanath (both BSc) (Int. Graduate Program in Medical Neurosciences) as well as Alexander Lucius (Medicine) during their lab practice. References Bandell, M., Dubin, A.E., Petrus, M.J., Orth, A., Mathur, J., Hwang, S.W., Patapoutian, A., 2006. High-throughput random mutagenesis screen reveals TRPM8 residues specifically required for activation by menthol. Nat. Neurosci. 9, 493e500. Bautista, D.M., Siemens, J., Glazer, J.M., Tsuruda, P.R., Basbaum, A.I., Stucky, C.L., Jordt, S.E., Julius, D., 2007. The menthol receptor TRPM8 is the principal detector of environmental cold. Nature 448, 204e208. Bednarz, J., Teifel, M., Friedl, P., Engelmann, K., 2000. Immortalization of human corneal endothelial cells using electroporation protocol optimized for human corneal endothelial and human retinal pigment epithelial cells. Acta Ophthalmol. Scand. 78, 130e136. Bednarz, J., Weich, H.A., Rodokanaki-von Schrenck, A., Engelmann, K., 1995. Expression of genes coding growth factors and growth factor receptors in differentiated and dedifferentiated human corneal endothelial cells. Cornea 14, 372e381. Behrendt, H.J., Germann, T., Gillen, C., Hatt, H., Jostock, R., 2004. Characterization of the mouse cold-menthol receptor TRPM8 and vanilloid receptor type-1 VR1 using a fluorometric imaging plate reader (FLIPR) assay. Br. J. Pharmacol. 141, 737e745. Bonanno, J.A., 2012. Molecular mechanisms underlying the corneal endothelial pump. Exp. Eye Res. 95, 2e7. Bruggemann, A., Farre, C., Haarmann, C., Haythornthwaite, A., Kreir, M., Stoelzle, S., George, M., Fertig, N., 2008. Planar patch clamp: advances in electrophysiology. Methods Mol. Biol. 491, 165e176. Bruggemann, A., Stoelzle, S., George, M., Behrends, J.C., Fertig, N., 2006. Microchip technology for automated and parallel patch-clamp recording. Small 2, 840e 846.

S. Mergler et al. / Experimental Eye Research 116 (2013) 337e349 Chuang, H.H., Neuhausser, W.M., Julius, D., 2004. The super-cooling agent icilin reveals a mechanism of coincidence detection by a temperature-sensitive TRP channel. Neuron 43, 859e869. Crawford, D.C., Moulder, K.L., Gereau, R.W., Story, G.M., Mennerick, S., 2009. Comparative effects of heterologous TRPV1 and TRPM8 expression in rat hippocampal neurons. PLoS. ONE 4, e8166. Engelmann, K., Bohnke, M., Friedl, P., 1988. Isolation and long-term cultivation of human corneal endothelial cells. Invest. Ophthalmol. Vis. Sci. 29, 1656e1662. Fonfria, E., Murdock, P.R., Cusdin, F.S., Benham, C.D., Kelsell, R.E., McNulty, S., 2006. Tissue distribution profiles of the human TRPM cation channel family. J. Recept. Signal. Transduct. Res. 26, 159e178. Grynkiewicz, G., Poenie, M., Tsien, R.Y., 1985. A new generation of Ca2þ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260, 3440e3450. Hirata, H., Oshinsky, M.L., 2012. Ocular dryness excites two classes of corneal afferent neurons implicated in basal tearing in rats: involvement of transient receptor potential channels. J. Neurophysiol. 107, 1199e1209. Kim, S.H., Nam, J.H., Park, E.J., Kim, B.J., Kim, S.J., So, I., Jeon, J.H., 2009. Menthol regulates TRPM8-independent processes in PC-3 prostate cancer cells. Biochim. Biophys. Acta 1792, 33e38. Kobayashi, K., Fukuoka, T., Obata, K., Yamanaka, H., Dai, Y., Tokunaga, A., Noguchi, K., 2005. Distinct expression of TRPM8, TRPA1, and TRPV1 mRNAs in rat primary afferent neurons with adelta/c-fibers and colocalization with trk receptors. J. Comp. Neurol. 493, 596e606. Kochukov, M.Y., McNearney, T.A., Fu, Y., Westlund, K.N., 2006. Thermosensitive TRP ion channels mediate cytosolic calcium response in human synoviocytes. Am. J. Physiol. Cell. Physiol. 291, C424eC432. Mahieu, F., Owsianik, G., Verbert, L., Janssens, A., De, S.H., Nilius, B., Voets, T., 2007. TRPM8-independent menthol-induced Ca2þ release from endoplasmic reticulum and Golgi. J. Biol. Chem. 282, 3325e3336. Malkia, A., Pertusa, M., Fernandez-Ballester, G., Ferrer-Montiel, A., Viana, F., 2009. Differential role of the menthol-binding residue Y745 in the antagonism of thermally gated TRPM8 channels. Mol. Pain 5, 62. McKemy, D.D., 2005. How cold is it? TRPM8 and TRPA1 in the molecular logic of cold sensation. Mol. Pain 1, 16. McKemy, D.D., Neuhausser, W.M., Julius, D., 2002. Identification of a cold receptor reveals a general role for TRP channels in thermosensation. Nature 416, 52e58. Mergler, S., Cheng, Y., Skosyrsky, S., Garreis, F., Pietrzak, P., Kociok, N., Dwarakanath, A., Reinach, P.S., Kakkassery, V., 2012a. Altered calcium regulation by thermo-sensitive transient receptor potential channels in etoposideresistant WERI-Rb1 retinoblastoma cells. Exp. Eye Res. 94, 157e173. Mergler, S., Dannowski, H., Bednarz, J., Engelmann, K., Hartmann, C., Pleyer, U., 2003. Calcium influx induced by activation of receptor tyrosine kinases in SV40transfected human corneal endothelial cells. Exp. Eye Res. 77, 485e495. Mergler, S., Pleyer, U., Reinach, P., Bednarz, J., Dannowski, H., Engelmann, K., Hartmann, C., Yousif, T., 2005. EGF suppresses hydrogen peroxide induced Ca2þ influx by inhibiting L-type channel activity in cultured human corneal endothelial cells. Exp. Eye Res. 80, 285e293. Mergler, S., Skrzypski, M., Sassek, M., Pietrzak, P., Pucci, C., Wiedenmann, B., Strowski, M.Z., 2012b. Thermo-sensitive transient receptor potential vanilloid channel-1 regulates intracellular calcium and triggers chromogranin A secretion in pancreatic neuroendocrine BON-1 tumor cells. Cell Signal 24, 233e246. Mergler, S., Strowski, M.Z., Kaiser, S., Plath, T., Giesecke, Y., Neumann, M., Hosokawa, H., Kobayashi, S., Langrehr, J., Neuhaus, P., Plockinger, U., Wiedenmann, B., Grotzinger, C., 2007. Transient receptor potential channel

349

TRPM8 agonists stimulate calcium influx and neurotensin secretion in neuroendocrine tumor cells. Neuroendocrinology 85, 81e92. Mergler, S., Valtink, M., Coulson-Thomas, V.J., Lindemann, D., Reinach, P.S., Engelmann, K., Pleyer, U., 2010. TRPV channels mediate temperature-sensing in human corneal endothelial cells. Exp. Eye Res. 90, 758e770. Milligan, C.J., Li, J., Sukumar, P., Majeed, Y., Dallas, M.L., English, A., Emery, P., Porter, K.E., Smith, A.M., McFadzean, I., Beccano-Kelly, D., Bahnasi, Y., Cheong, A., Naylor, J., Zeng, F., Liu, X., Gamper, N., Jiang, L.H., Pearson, H.A., Peers, C., Robertson, B., Beech, D.J., 2009. Robotic multiwell planar patch-clamp for native and primary mammalian cells. Nat. Protoc. 4, 244e255. Pan, Z., Yang, H., Reinach, P.S., 2011. Transient receptor potential (TRP) gene superfamily encoding cation channels. Hum. Genom. 5, 108e116. Parra, A., Madrid, R., Echevarria, D., del, O.S., Morenilla-Palao, C., Acosta, M.C., Gallar, J., Dhaka, A., Viana, F., Belmonte, C., 2010. Ocular surface wetness is regulated by TRPM8-dependent cold thermoreceptors of the cornea. Nat. Med. 16, 1396e1399. Peier, A.M., Moqrich, A., Hergarden, A.C., Reeve, A.J., Andersson, D.A., Story, G.M., Earley, T.J., Dragoni, I., McIntyre, P., Bevan, S., Patapoutian, A., 2002a. A TRP channel that senses cold stimuli and menthol. Cell 108, 705e715. Peier, A.M., Reeve, A.J., Andersson, D.A., Moqrich, A., Earley, T.J., Hergarden, A.C., Story, G.M., Colley, S., Hogenesch, J.B., McIntyre, P., Bevan, S., Patapoutian, A., 2002b. A heat-sensitive TRP channel expressed in keratinocytes. Science 296, 2046e2049. Rawls, S.M., Gomez, T., Ding, Z., Raffa, R.B., 2007. Differential behavioral effect of the TRPM8/TRPA1 channel agonist icilin (AG-3-5). Eur. J. Pharmacol. 575, 103e104. Sandboe, F.D., Medin, W., Froslie, K.F., 2003. Influence of temperature on corneas stored in culture medium. A comparative study using functional and morphological methods. Acta Ophthalmol. Scand 81, 54e59. Story, G.M., Peier, A.M., Reeve, A.J., Eid, S.R., Mosbacher, J., Hricik, T.R., Earley, T.J., Hergarden, A.C., Andersson, D.A., Hwang, S.W., McIntyre, P., Jegla, T., Bevan, S., Patapoutian, A., 2003. ANKTM1, a TRP-like channel expressed in nociceptive neurons, is activated by cold temperatures. Cell 112, 819e829. Tominaga, M., Caterina, M.J., 2004. Thermosensation and pain. J. Neurobiol. 61, 3e 12. Valero, M., Morenilla-Palao, C., Belmonte, C., Viana, F., 2011. Pharmacological and functional properties of TRPM8 channels in prostate tumor cells. Pflugers Arch. 461, 99e114. Valtink, M., Gruschwitz, R., Funk, R.H., Engelmann, K., 2008. Two clonal cell lines of immortalized human corneal endothelial cells show either differentiated or precursor cell characteristics. Cells Tissues. Organs 187, 286e294. Voets, T., Droogmans, G., Wissenbach, U., Janssens, A., Flockerzi, V., Nilius, B., 2004. The principle of temperature-dependent gating in cold- and heat-sensitive TRP channels. Nature 430, 748e754. Voets, T., Owsianik, G., Nilius, B., 2007. TRPM8. Handb. Exp. Pharmacol., 329e344. Vriens, J., Appendino, G., Nilius, B., 2009. Pharmacology of vanilloid transient receptor potential cation channels. Mol. Pharmacol. 75, 1262e1279. Weil, A., Moore, S.E., Waite, N.J., Randall, A., Gunthorpe, M.J., 2005. Conservation of functional and pharmacological properties in the distantly related temperature sensors TRVP1 and TRPM8. Mol. Pharmacol. 68, 518e527. Xing, H., Chen, M., Ling, J., Tan, W., Gu, J.G., 2007. TRPM8 mechanism of cold allodynia after chronic nerve injury. J. Neurosci. 27, 13680e13690. Zhang, L., Barritt, G.J., 2004. Evidence that TRPM8 is an androgen-dependent Ca2þ channel required for the survival of prostate cancer cells. Cancer Res. 64, 8365e 8373.