November 2010
Volume 51, Issue 11
Free
Retinal Cell Biology  |   November 2010
Heat-Sensitive TRPV Channels in Retinal Pigment Epithelial Cells: Regulation of VEGF-A Secretion
Author Affiliations & Notes
  • Sönke Cordeiro
    From the Experimentelle Ophthalmologie, Klinik und Poliklinik für Augenheilkunde, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany;
    Institut für Neurophysiologie, Medizinische Hochschule Hannover, Hannover, Germany; and
  • Sebastian Seyler
    From the Experimentelle Ophthalmologie, Klinik und Poliklinik für Augenheilkunde, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany;
  • Julia Stindl
    Experimentelle Ophthalmologie, Klinik und Poliklinik für Augenheilkunde, University Health Center Regensburg, Regensburg, Germany.
  • Vladimir M. Milenkovic
    Experimentelle Ophthalmologie, Klinik und Poliklinik für Augenheilkunde, University Health Center Regensburg, Regensburg, Germany.
  • Olaf Strauss
    From the Experimentelle Ophthalmologie, Klinik und Poliklinik für Augenheilkunde, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany;
    Experimentelle Ophthalmologie, Klinik und Poliklinik für Augenheilkunde, University Health Center Regensburg, Regensburg, Germany.
  • Corresponding author: Olaf Strauss, Experimentelle Ophthalmologie, Klinik und Poliklinik fuer Augenheilkunde, Klinikum der Universität Regensburg, Franz-Josef-Strauss Allee 11, 93053 Regensburg, Germany; strauss@eye-regensburg.de
Investigative Ophthalmology & Visual Science November 2010, Vol.51, 6001-6008. doi:10.1167/iovs.09-4720
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Sönke Cordeiro, Sebastian Seyler, Julia Stindl, Vladimir M. Milenkovic, Olaf Strauss; Heat-Sensitive TRPV Channels in Retinal Pigment Epithelial Cells: Regulation of VEGF-A Secretion. Invest. Ophthalmol. Vis. Sci. 2010;51(11):6001-6008. doi: 10.1167/iovs.09-4720.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose.: Choroidal neovascularization in age-related macular degeneration is caused, to a large extent, by increased secretion of vascular endothelial growth factor (VEGF)-A by the retinal pigment epithelium (RPE). The purpose of the study was to identify pathways that lead to increased VEGF secretion by the RPE.

Methods.: Ca2+ signaling was studied in ARPE-19 and human RPE cells in primary culture by means of Ca2+ imaging. Membrane conductance was measured in the whole-cell configuration of the patch-clamp technique. VEGF-A secretion was measured by using ELISA.

Results.: Freshly isolated RPE cells or ARPE-19 cells were shown to express TRPV1, -2, -3, and -4 channels. Increasing the temperature or stimulation by IGF-1 increased the VEGF-A secretion rate in both cell types. These effects were both sensitive to the TRPV channel blocker ruthenium red (20 μM). The heat-inducible Ca2+ signals were blocked by the TRPV channel blockers La3+ and ruthenium red by 68% and 52%, respectively. In contrast, high concentrations of 2-APB (3 mM) increased [Ca2+]i, whereas the TRPV1 channel opener capsaicin and the TRPV3 channel opener camphor had no effect. Reduction of TRPV2 expression by siRNA attenuated the heat-evoked Ca2+ response. In addition, a heat-activated inwardly rectifying current was measured that was completely blocked by ruthenium red. IGF-1 also increased whole-cell current with a corresponding increase in [Ca2+]i, which was blocked by the PI3-kinase blocker LY294002.

Conclusions.: The data strongly suggest that TRPV2 channels expressed by the RPE are involved in the Ca2+ signaling that mediates both heat-dependent and IGF-1 (via PI3-kinase activation)-induced VEGF secretion.

Age-related macular degeneration (AMD) is the main cause of vision loss in industrialized nations. 1 4 The main complication in AMD is the formation of new blood vessels growing from the choroid into the retina, which can subsequently lead to intraocular bleeding and scar formation. This wet form of AMD accounts for only 10% to 20% of overall AMD. However, this subtype with choroidal neovascularization (CNV) is responsible for approximately 90% of severe vision loss. 5,6 A major factor inducing the formation of new blood vessels is vascular endothelial growth factor (VEGF)-A. 7 Neutralization of VEGF-A has provided the first AMD therapy resulting in vision improvement. 8 Although these anti-VEGF therapies are very efficacious in preventing vision loss, there is still reoccurrence of vessel growth due to constitutive VEGF-A expression in the eye, which requires repeated treatments with the risk of vision loss in long-term treatments. 9 In improving anti–VEGF-A strategies, developers of advanced therapies must consider the network of growth factors that underlie increased VEGF-A production. 9 The major source of angiogenic (e.g., VEGF) and antiangiogenic (e.g., pigment epithelial derived factor; PEDF) factors that play a role in the modulation and progression of CNV is the RPE. 10,11 In addition, the RPE has been shown to express a variety of cytokine receptors. 12 14 The specific activation of some of these cytokine receptors by their ligands has been associated with the regulation of VEGF secretion in the RPE. 15 19 Although it is well known that these factors may induce VEGF secretion, the intracellular pathways leading to this VEGF secretion remain only poorly understood. Insulin-like growth factor (IGF)-1 plays a well-known role in the induction of neovascularization in the retina. 20,21 More recently, it has been shown that IGF-1 stimulates VEGF secretion by the RPE. 22 24 In addition, the VEGF secretion by the RPE has been shown to depend on elevations of the intracellular free Ca2+ concentration ([Ca2+]i). 22 Rosenthal et al., 22 suggested that IGF-1 may function as a rescue signal from injured photoreceptors before the onset of CNV induction. This notion is based on IGF-1 expression by photoreceptors in AMD retinas before CNV development. 
IGF-1 has been shown to activate the transient receptor potential channels TRPV1 and -2 in the heterologous expression system. 25,26 The RPE is known to express a couple of Ca2+ channels, 27 but to date, nothing is known about the expression of TRPV channels by the RPE. Since IGF-1–dependent secretion of VEGF depends on increases in intracellular free Ca2+, it is likely that IGF-1–dependent activation of TRPV channels may contribute to this regulatory process. 
The purpose of this study was to elucidate the intracellular signaling in the RPE that induces an enhanced VEGF secretion. That signaling led us to the TRPV currents in the RPE carried by TRPV2 channels. The activation of these channels either by heat stimuli or by IGF-1 caused an increased VEGF secretion. 
Materials and Methods
Cell Culture
The human RPE cell line ARPE-19 was cultured in Dulbecco's modified Eagle's medium-F-12 nutrient mixture containing 10% fetal bovine serum, insulin-transferrin-sodium (Roche Diagnostics, Mannheim, Germany), nonessential amino acids, and penicillin/streptomycin at 37°C in a humidified ambient atmosphere containing 5% CO2. They were passaged twice a week. For Fura-2 measurements, the cells were seeded on coverslips and cultured to confluence. 
Primary cultures of human RPE (hRPE) cells were used. The anterior part of human donor eyes, including the vitreous and retina were removed. The RPE with the choroid were carefully separated from the sclera, washed with PBS, and incubated overnight with collagenase IA/IV (0.5 mg/mL each) in serum-free culture medium. The dissociated cells were collected by centrifugation (50g, 5 minutes) and cultured on coverslips in the same medium as the ARPE-19 cells. These still-pigmented RPE cells were used for Ca2+ measurements after they reached confluence. For patch-clamp measurements, the same cells were used in the first week after preparation. 
The protocol adhered to the tenets of the Declaration of Helsinki for research involving human tissue. 
RNA Isolation and RT-PCR
Human RPE was obtained from organ donors within 24 hours of death. After the cornea was removed for transplantation, the eyes were dissected for RPE preparation. The anterior parts of the eyes including the vitreous and the retina were removed. The posterior part was rinsed with ice cold PBS (without Ca2+ and Mg2+) to wash away residuals of the neuronal retina. With a fine pair of forceps, the RPE was gently brushed away. The RPE cells were collected and lysed (RNeasy Mini Kit; Qiagen, Hilden, Germany). Total RNA from ARPE-19 cells was prepared from confluent cultures grown in a 25-cm2 culture flask. The RNA was isolated (RNeasy Mini kit; Qiagen), according to the manufacturer's instructions, and 1 μg was reverse transcribed at 37°C for 1 hour in the following reaction mixture: 1 μg oligo dT primer (Invitrogen, Karlsruhe, Germany), 1 mM of each dNTP, 20 U RNA stabilizer (RNAguard; GE Health Care, Freiburg, Germany), and 20 U M-MLV reverse transcriptase (Invitrogen). For control PCR reactions, human total brain RNA (Stratagene, Heidelberg, Germany) was reverse transcribed under the same conditions. PCR experiments were performed with 1 μL cDNA in 50-μL PCR reaction mixtures with Taq DNA polymerase (Stratagene) and 1.5 picomoles of sense and antisense oligonucleotides specific for the different TRPV channel subunits (Table 1). The identity of the amplification products was confirmed by sequencing. 
Table 1.
 
Oligonucleotides Used for the Detection of TRPV Channel Subunits
Table 1.
 
Oligonucleotides Used for the Detection of TRPV Channel Subunits
Protein (Accession No.*) Oligonucleotide Sequences Annealing Temp. Degrees (bp)
TRPV1 (NM_080704) Forward 5′-GCCTGGAGCTGTTCAAGTTC-3′ 59 (177)
Reverse 5′-TCTCCTGTGCGATCTTGTTG-3′
TRPV2 (NM_016113) Forward 5′-CAAACCGATTTGACCGAGAT-3′ 59 (167)
Reverse 5′-GTTCAGCACAGCCTTCATCA-3′
TRPV3 (NM_145068) Forward 5′-ACGAGGCAACAACATCCTTC-3′ 56 (226)
Reverse 5′-CCGCTTCTCCTTGATCTCAC-3′
TRPV4 (NM_147204) Forward 5′-GACGGGGACCTATAGCATCA-3′ 59 (228)
Reverse 5′-AACAGGTCCAGGAGGAAGGT-3′
TRPV5 (NM_019841) Forward 5′-GGAGCTTGTGGTCTCCTCTG-3′ 59 (185)
Reverse 5′-GAAACTTAAGGGGGCGGTAG-3′
TRPV6 (NM_018646) Forward 5′-GCCTATGGAGCAAGTTCTGC-3′ 50 (242)
Reverse 5′-GGCCTCCAGGTTGTCATAGA-3′
Real-Time Quantitative qPCR
ARPE-19 cells were collected and subjected to homogenization and mRNA isolation. Total mRNA was isolated by using (NucleoSpin RNAII kit; Macherey-Nagel GmbH, Düren, Germany). One microgram of total RNA was reverse transcribed to cDNA (I-Script cDNA synthesis kit; Bio-Rad, Munich, Germany). For quantification, a SYBR green-based kit (2× SSO Fast EvaGreen kit; Bio-Rad) was used. Real-time PCR reactions were performed in a 20-μL reaction volume, and all reactions were prepared in triplicate. Quantitative real-time RT-PCR was performed on a thermal cycler (MJ Research Opticon Eclipse Real-Time Cycler; Bio-Rad). Oligonucleotide primer pairs spanning an exon–exon boundary used in the study are listed in Table 1. Normalization of gene expression was achieved according to the method described by Vandesompele et al. 28 β-Actin was used as a housekeeping gene for the calculation normalization factor in each tissue. To verify specificity of PCR amplification, dissociation curves were generated by gradually increasing the temperature from 60°C to 94°C by ramping it up 0.5°C every 10 seconds. 
Small Interfering RNA
A lipophilic transfection reagent (Lipofectamine 2000; Invitrogen) was used, according to the manufacturer's recommendation, to transfect the ARPE-19 cells with TRPV2 siRNA, which contained a mixture of four siRNAs and scrambled siRNA (Thermo Scientific, Karlsruhe, Germany). 
Measurement of Intracellular Free Ca2+ Concentrations
The hRPE primary cultures or ARPE-19 cells grown on coverslips to confluence were washed with Ringer solution (130 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 5 mM glucose, 10 mM HEPES [pH 7.3] with NaOH) and loaded with fura-2-AM ester (Fluka, AG, Buchs, Switzerland) for 40 minutes in the dark at room temperature in Ringer solution containing 10 μM Fura-2. The cells were washed and after that incubated for at least 30 minutes with Ringer solution. The coverslips were placed into a bath chamber perfused constantly with Ringer solution and mounted onto an inverted microscope (Axiovert 35; Carl Zeiss Meditec, Oberkochen, Germany) equipped with a 40× objective (Fluar; Carl Zeiss Meditec). To exclude the possible stimulation of mechanosensitive channels by the perfusion system, we stopped the perfusion and found no change in [Ca2+]i. We performed ratiometric measurements of Fura-2 fluorescence at 5-second intervals, with a high-speed polychromator (Visitron Systems, Puchheim, Germany), alternating the wavelength of excitation light between 340 and 380 nm. Emitted light was filtered with a 510-nm filter and detected by a cooled charged-coupled device camera (CoolSNAP; Roper Scientific, Duluth, GA). Data were collected (MetaFlour software) and analyzed (MetaAnalysis software; Universal Imaging Corp., West Chester, PA). Intracellular free Ca2+ ([Ca2+]i) was measured by superfusing the cells with Ca2+-free Ringer solution supplemented with 1 mM EGTA and 1 μM ionomycin. The cells were then superfused with Ringer solution supplemented with 1 μM ionomycin and saturating Ca2+ concentration. [Ca2+]i was calculated according to Grynkiewicz et al. 29 :   where R is the fluorescence intensity ratio (F (340)/F ( 380)), R m i n is the minimum fluorescence ratio (with EGTA), R max is the maximum ratio (with saturating Ca2+ and ionomycin), K d is the dissociation constant of Fura-2, and S f and S b are the maximum and minimum fluorescence, respectively, after excitation with 380 nm. 
VEGF Secretion by the RPE
Before the experiment was started, confluent ARPE-19 cultures were held 24 hours in serum-free medium. The medium was exchanged to fresh serum-free medium again. The cells were then stimulated, by either a 5-minute incubation at 56°C or by IGF-1 (50 ng/mL). The concentration of VEGF-A (VEGF-165) secreted into the medium was measured 30 minutes after the heat stimulus or 4 hours after the IGF-1 stimulation by ELISA (Biosource International, Solingen, Germany) according to the manufacturer's instructions. The VEGF-A concentrations were measured from normalized 105 cells. The cells were counted at the end of the experiment. In experiments using heat stimulation, the number of cells was not significantly reduced. 
Patch-Clamp Measurements
RPE cells from human donor eyes or ARPE-19 cells were placed in a bath chamber on the stage of an inverted microscope. The bath solution was composed of (in mM) 130 NaCl, 5 KCl, 2 MgCl2, 2 CaCl2, 10 HEPES, and 5 glucose, adjusted to pH 7.3 with NaOH. Patch-clamp electrodes with a resistance of 3 to 5 MΩ were pulled from borosilicate glass (DMZ Universal Puller; Dagan Corp., Minneapolis, MN). The electrodes were filled with a solution containing (in mM) 140 KCl, 2 MgCl2, 1 CaCl2, 2.5 EGTA, and 10 HEPES, adjusted to pH 7.3 with KOH. Whole-cell currents were measured with a patch-clamp amplifier (EPC-9; HEKA Electronik, GmbH, Lambrecht/Pfalz, Germany) in conjunction with data acquisition and analysis software (TIDA; HEKA Electronik, GmbH). Fast- and slow-capacity transients were compensated for and series resistance errors were compensated to at least 75%. All experiments were performed at room temperature (22–25°C). 
Data Analysis
Statistical significance was tested by using one-way analysis of variance (ANOVA). All data are expressed as the mean ± SEM. 
Results
Effect of Heat on Secretion of VEGF-A by the RPE
RT-PCR-based analysis of the expression profile of TRPV channels in freshly isolated hRPE cells showed the expression of TRPV1, -2, -3, and -4 (Fig. 1A, left). The same expression profile was observed in ARPE-19 cells (Fig. 1A, right). We have shown previously that IGF-1 induces increased VEGF secretion accompanied by a sustained increase of [Ca2+]i in the RPE. 22 Furthermore, it has been shown in a heterologous expression system that IGF-1 can activate heat-sensitive TRPV channels. 25 To test whether IGF-1–induced VEGF-A secretion involves the activity of TRPV channels, IGF-1–induced VEGF-A secretion was investigated in the presence of the TRPV channel blocker ruthenium red (20 μM). IGF-1 induced a 70% increase in basic VEGF-A secretion, whereas in the presence of ruthenium red, it increased VEGF-A secretion by only 40%. To test whether the activation of TRPV channels can increase VEGF-A secretion, we measured VEGF secretion after heat stimulation of ARPE-19 cells. First, the cells were serum-starved for 24 hours, and, after replacement of the medium with fresh serum-free medium, a 56°C heat stimulus was applied to the cells for 5 minutes. After 30 minutes, the VEGF secreted by the ARPE-19 cells increased by 263% ± 32% compared with cells without heat stimulus. This VEGF secretion was significantly reduced to an increase of only 114% ± 30% by the addition of the TRPV blocker ruthenium red (20 μM) before application of the heat stimulus (Fig. 1). 
Figure 1.
 
Expression of TRPV channels in the human RPE and VEGF secretion by RPE cells. (A) Left: expression profile of TRPV channels in freshly isolated hRPE cells. Right: expression profile of TRPV channels in ARPE-19 cells. (B) IGF-1 induced secretion of VEGF-A by ARPE-19 cells. Shown is the normalized VEGF concentration in the medium. After stimulation by IGF-1 (50 ng/mL) and an additional 4-hour incubation at 37°C, the VEGF concentration in the medium was determined by ELISA. The values were normalized to control measurements without heat stimulus. In the presence of ruthenium red (20 μM), the IGF-1–induced VEGF secretion is decreased (n = 3). (C) Heat-induced secretion of VEGF-A by ARPE-19 cells. The normalized VEGF concentration in the medium is shown. Then, after a 5-minute heat-stimulus to 56°C and an additional 30-minute incubation at 37°C, the VEGF concentration in the medium was determined by ELISA. The values were normalized to control measurements without heat stimulus. The heat stimulation in the presence of ruthenium red (20 μM) clearly suppressed the increased VEGF secretion (n = 3). (*P < 0.05).
Figure 1.
 
Expression of TRPV channels in the human RPE and VEGF secretion by RPE cells. (A) Left: expression profile of TRPV channels in freshly isolated hRPE cells. Right: expression profile of TRPV channels in ARPE-19 cells. (B) IGF-1 induced secretion of VEGF-A by ARPE-19 cells. Shown is the normalized VEGF concentration in the medium. After stimulation by IGF-1 (50 ng/mL) and an additional 4-hour incubation at 37°C, the VEGF concentration in the medium was determined by ELISA. The values were normalized to control measurements without heat stimulus. In the presence of ruthenium red (20 μM), the IGF-1–induced VEGF secretion is decreased (n = 3). (C) Heat-induced secretion of VEGF-A by ARPE-19 cells. The normalized VEGF concentration in the medium is shown. Then, after a 5-minute heat-stimulus to 56°C and an additional 30-minute incubation at 37°C, the VEGF concentration in the medium was determined by ELISA. The values were normalized to control measurements without heat stimulus. The heat stimulation in the presence of ruthenium red (20 μM) clearly suppressed the increased VEGF secretion (n = 3). (*P < 0.05).
Heat-Inducible Channels in the RPE
To show the functional presence of TRPV channels in RPE cells, we measured [Ca2+]i by using the Ca2+-sensitive fluorescence dye Fura-2 in ARPE-19 cells (Fig. 2A) and in hRPE cells in primary culture (Fig. 2B). Increasing the temperature of the bath of RPE cells in primary culture from room temperature to 56°C resulted in an increase in [Ca2+]i that lasted 16 ± 4.75 seconds (n = 12). The maximum [Ca2+]i increase was to 169% ± 15%, compared with baseline (n = 12). A comparable observation was made in the ARPE-19 cells. Increases in the temperature to 56°C transiently increased [Ca2+]i to 177% ± 8%, compared with baseline (n = 34). This increase in intracellular Ca2+ lasted for 34 ± 5.52 seconds (n = 34). Since relatively high temperatures were needed to evoke heat-induced Ca2+ transients, the possible involvement of TRPV2 channels was investigated by using a siRNA approach. Treatment of ARPE-19 cells with siRNA against TRPV2 reduced the TRPV2 expression measured by qPCR to 21% ± 4% of control (unspecific siRNA, n = 3; P = 0.0033). In cells that have been treated by siRNA against TRPV2, the amplitude of the heat-evoked Ca2+ transients was reduced to 28% ± 2% of the mean heat-induced Ca2+ increase (n = 14; P = 0.0001). 
Figure 2.
 
Heat evoked increases in [Ca2+]i. Increasing the bath temperature from room temperature to 56°C led to an increase in [Ca2+]i in (A) ARPE-19 and (B) hRPE cells. (C) Downregulation of TRPV2 expression in ARPE-19 cells by siRNA: treatment of RPE cells with siRNA against TRPV2 decreased TRPV2 expression compared with cells treated with scrambled siRNA (n = 3 each group). (D) Heat-induced Ca2+ transients in ARPE-19 cells treated with siRNA against TRPV2 (n = 12 scrambled siRNA; n = 14 siRNA; **P < 0.01; ***P < 0.0001).
Figure 2.
 
Heat evoked increases in [Ca2+]i. Increasing the bath temperature from room temperature to 56°C led to an increase in [Ca2+]i in (A) ARPE-19 and (B) hRPE cells. (C) Downregulation of TRPV2 expression in ARPE-19 cells by siRNA: treatment of RPE cells with siRNA against TRPV2 decreased TRPV2 expression compared with cells treated with scrambled siRNA (n = 3 each group). (D) Heat-induced Ca2+ transients in ARPE-19 cells treated with siRNA against TRPV2 (n = 12 scrambled siRNA; n = 14 siRNA; **P < 0.01; ***P < 0.0001).
These heat-evoked responses were sensitive to the TRPV blockers La3+ and ruthenium red (Fig. 3). To test the effects of these blockers, we exposed the cells to 56°C two times in succession with a 500-second pause between. The second heat exposure was in the presence of the respective blocker. In control experiments without blocker, the second peak did not differ in amplitude from the first peak (ARPE-19: second peak 90% ± 19% of the first peak, n = 10, P = 0.76; hRPE: second peak 93% ± 49% of the first peak, n = 3, P = 0.9). Increasing the temperature to 56°C in the presence of 100 μM La3+ resulted in a significantly lesser second increase in [Ca2+]i compared with the control measurement without La3+. This effect was observed in cells of the ARPE-19 cell line (control: increase by 57.51 ± 15 nM, n = 11; La3+: 37.3 ± 5 nM, n = 21) as well as in hRPE cells in primary culture (control: increase by 247 ± 72 nM, n = 9; La3+: 31.48 ± 7.9 nM, n = 10). This corresponds to a reduction by 26% ± 9% and by 68% ± 6%, respectively (Fig. 3E). We used the same protocol with two successive increases in temperature to investigate the effect of ruthenium red. In the presence of 1 μM ruthenium red, the second heat-evoked increase in intracellular Ca2+ was less than that without ruthenium red. The heat-evoked responses were reduced in both cells from the ARPE-19 cell line (control: increase by 91.4 ± 29 nM; n = 11 vs. ruthenium red: 36.61 ± 9 nM; n = 10) and in cells in primary culture (control: increase by 189.2 ± 91.09 nM; n = 3 vs. ruthenium red: 72.21 ± 49.15 nM; n = 5), corresponding to a reduction by 42% ± 11% and by 52% ± 13%, respectively (Fig. 3F). Thus, the heat-evoked responses in RPE cells show pharmacologic characteristics comparable to those of TRPV channels. To obtain more specific data about the subtype of the TRPV channel, which is active in RPE cells, several agonists for different TRPV channels were used. High concentrations of 2-APB (2-aminoethoxydiphenyl borate) are known to open TRPV1, -2, and -3 channels. 30 Application of 3 mM 2-APB resulted in increases in ARPE-19 cells of cytosolic free Ca2+ by 70.15 ± 5.12 nM (n = 3; Figs. 4A, 4D) and in hRPE cells in primary culture by 51.49 ± 7.92 nM (n = 3; Fig 4D). Application of the TRPV3 channel opener camphor 31 (400 μM; Fig. 4B) or the TRPV1 channel opener capsaicin 30 (1 μM; Fig. 4C) did not change the cytosolic free Ca2+ in ARPE-19 cells. 
Figure 3.
 
Effect of the TPRV channel blocker La3+ and ruthenium red in RPE cells. The effect of the blocker La3+ (100 μM) and ruthenium red (1 μM) were investigated by using two heat steps (increase from room temperature to 56°C) in succession: the first without blocker, and the second with blocker. Statistical tests were performed to compare the increase in Ca2+ with versus without blocker. Original recordings showing the effects of (A) La3+ (100 μM; bar) and (B) ruthenium red (1 μM; bar) on heat-evoked Ca2+ increases in ARPE-19 cells. Original recordings showing the effects of (C) La3+ (100 μM; bar) and (D) ruthenium red (1 μM; bar) on heat-evoked Ca2+ increases in hRPE cells in primary culture. Comparison of the second heat-evoked Ca2+ increase (as a percentage of the first one), without and with (E) La3+ and (F) ruthenium red in ARPE-19 cells as well as in hRPE cells in primary culture. In the presence of both La3+ and ruthenium red the heat-evoked Ca2+ increases were significantly reduced. (*P < 0.05).
Figure 3.
 
Effect of the TPRV channel blocker La3+ and ruthenium red in RPE cells. The effect of the blocker La3+ (100 μM) and ruthenium red (1 μM) were investigated by using two heat steps (increase from room temperature to 56°C) in succession: the first without blocker, and the second with blocker. Statistical tests were performed to compare the increase in Ca2+ with versus without blocker. Original recordings showing the effects of (A) La3+ (100 μM; bar) and (B) ruthenium red (1 μM; bar) on heat-evoked Ca2+ increases in ARPE-19 cells. Original recordings showing the effects of (C) La3+ (100 μM; bar) and (D) ruthenium red (1 μM; bar) on heat-evoked Ca2+ increases in hRPE cells in primary culture. Comparison of the second heat-evoked Ca2+ increase (as a percentage of the first one), without and with (E) La3+ and (F) ruthenium red in ARPE-19 cells as well as in hRPE cells in primary culture. In the presence of both La3+ and ruthenium red the heat-evoked Ca2+ increases were significantly reduced. (*P < 0.05).
Figure 4.
 
Further pharmacologic analysis of the effect of TRPV channel openers on [Ca2+]i in ARPE-19 cells. (A) High concentrations of the TRPV2 channel opener 2-APB (3 mM; bar) led to an increase in [Ca2+]i. (B) The TRPV3 opener camphor (400 μM ; bar) did not change the level of [Ca2+]i. (C) The TRPV1 opener capsaicin (1 μM; bar) did not change the level of [Ca2+]i. (D) Relative increases in [Ca2+]i in ARPE-19 cells or in RPE cells in primary culture by 3 mM 2-APB (n = 3 each group).
Figure 4.
 
Further pharmacologic analysis of the effect of TRPV channel openers on [Ca2+]i in ARPE-19 cells. (A) High concentrations of the TRPV2 channel opener 2-APB (3 mM; bar) led to an increase in [Ca2+]i. (B) The TRPV3 opener camphor (400 μM ; bar) did not change the level of [Ca2+]i. (C) The TRPV1 opener capsaicin (1 μM; bar) did not change the level of [Ca2+]i. (D) Relative increases in [Ca2+]i in ARPE-19 cells or in RPE cells in primary culture by 3 mM 2-APB (n = 3 each group).
To show that the heat-induced increase in intracellular Ca2+ is due to heat-dependent activation of Ca2+-conducting ion channels, we measured whole-cell currents in the RPE cells with the patch-clamp technique. In these experiments, temperature was raised to only 45°C because the membrane instability at 56°C makes whole-cell, patch-clamp recordings impossible at this temperature. In control measurements in ARPE-19 cells, however, a 45°C stimulus led to an increase in [Ca2+]i that was lower than that induced by the 56°C stimulus (Fig. 5A). Thus, increases of temperature to 45°C are also suitable to measure heat-evoked responses of membrane currents. To continuously monitor changes in the membrane conductance, we clamped the membrane potential of the cells to −40 mV which corresponds to the resting potential of RPE cells. From this holding potential, the cells were stimulated using repeated voltage-ramps between −140 and +60 mV. Increasing the temperature to 45°C resulted in a transient increase in the membrane conductance (Fig. 5A). This increase in the membrane conductance resulted in an increase in the inward current at −40 mV (control: −34.87 ± 10.4 pA; at 45°C: −57.5 ± 16.05 pA; n = 7; Fig. 5B) that corresponded to an increase in inward current at –40 mV of 67% ± 12% (n = 7). The inward current lasted for 10 seconds, a time comparable to that of heat-evoked increases in intracellular Ca2+. The currents showed a reversal potential of 0 mV, indicating the activation of a nonselective cation channel. The heat-evoked currents were sensitive to ruthenium red. In the presence of 20 μM ruthenium red an increase of the temperature to 45°C failed to activate inward currents (Fig. 5C). Analysis of the currents at different potentials showed that only the peak currents at −40 and −140 mV were blocked by ruthenium red and not the membrane currents at +60 mV, indicating the activation of another outwardly rectifying current. Thus, heat led to the activation of inwardly rectifying cation channels. 
Figure 5.
 
Whole-cell recordings of heat-evoked membrane currents. (A) Changes in intracellular free Ca2+ by temperature increase to 45°C. After a recovery time of 25 minutes, the cells showed a full response to an increase to 56°C, as well. (B) Recording of the whole-cell recording of membrane currents. The membrane conductance potential was read by clamping to −40 mV, and the cell was electrically stimulated by voltage-ramps between −140 and +60 mV every 1.5 seconds. Left: recording showing the effect of increasing the bath temperature from room temperature to +45°C (arrow). Shortly after the temperature was increased, the membrane conductance increased. First, a transient inwardly rectifying conductance was observed that was followed by the sustained activation of a nonrectifying conductance, which remained sustained. Middle: recording of the whole-cell currents at −40 mV holding potential showing the fast transient heat-evoked (arrow) inward current. Right: comparison of whole-cell currents elicited by ramped voltages before and during an increase in the temperature to 45°C. (C) Recording of the whole-cell membrane currents in the presence of ruthenium red (20 μM). To monitor the membrane conductance the membrane potential was clamped to −40 mV and the cell was electrically stimulated by ramped voltages between −140 and +60 mV with increases every 1.5 seconds. Left: the effect of increasing the bath temperature from room temperature to +45°C (arrow). In the presence of ruthenium red, increasing the temperature failed to induce the transient inward current. Middle: recording of the whole-cell currents only at −40 mV holding potential in the presence of ruthenium red showing that increasing the temperature to 45°C failed to activate the transient inward current. Right: comparison of whole-cell currents elicited by ramped voltages, before and during an increase from room temperature to 45°C in the presence of ruthenium red.
Figure 5.
 
Whole-cell recordings of heat-evoked membrane currents. (A) Changes in intracellular free Ca2+ by temperature increase to 45°C. After a recovery time of 25 minutes, the cells showed a full response to an increase to 56°C, as well. (B) Recording of the whole-cell recording of membrane currents. The membrane conductance potential was read by clamping to −40 mV, and the cell was electrically stimulated by voltage-ramps between −140 and +60 mV every 1.5 seconds. Left: recording showing the effect of increasing the bath temperature from room temperature to +45°C (arrow). Shortly after the temperature was increased, the membrane conductance increased. First, a transient inwardly rectifying conductance was observed that was followed by the sustained activation of a nonrectifying conductance, which remained sustained. Middle: recording of the whole-cell currents at −40 mV holding potential showing the fast transient heat-evoked (arrow) inward current. Right: comparison of whole-cell currents elicited by ramped voltages before and during an increase in the temperature to 45°C. (C) Recording of the whole-cell membrane currents in the presence of ruthenium red (20 μM). To monitor the membrane conductance the membrane potential was clamped to −40 mV and the cell was electrically stimulated by ramped voltages between −140 and +60 mV with increases every 1.5 seconds. Left: the effect of increasing the bath temperature from room temperature to +45°C (arrow). In the presence of ruthenium red, increasing the temperature failed to induce the transient inward current. Middle: recording of the whole-cell currents only at −40 mV holding potential in the presence of ruthenium red showing that increasing the temperature to 45°C failed to activate the transient inward current. Right: comparison of whole-cell currents elicited by ramped voltages, before and during an increase from room temperature to 45°C in the presence of ruthenium red.
IGF-1 Activation of TRPV Channels in the RPE
To show that a comparable changed ion conductance is responsible for the well-known IGF-1–dependent increase in VEGF secretion, we applied IGF-1 to the bath solution while we measured the cells by the patch-clamp technique. The cells were kept at a holding potential of −40 mV and repeatedly stimulated by ramping from −140 to +60 mV. In three of four cells tested, the addition of IGF-1 led to a dramatic increase in membrane conductance of the RPE cells (Fig. 6A). The current increase occurred a rather long time after the time point of IGF-1 application: 536.67 ± 39.3 seconds after application of IGF-1 (Fig. 6A, arrow). In mock-treated cells (IGF-1 buffer without IGF-1) no change in the membrane conductance was observed (Fig. 6B). As after heat stimulation, the application of IGF-1 predominantly led to the activation of inward currents in ARPE-19 cells (the increase at −140 mV was up to 100-fold, whereas the current increase at +60 mV was only 2-fold). The IGF-1–stimulated increase in membrane conductance was completely blocked by La3+ (100 μM; Fig. 6C). At the time point of the strongest activation of inward currents, the reversal potential shifted by 8.83 ± 2.48 mV to more positive potentials (Fig. 6D; from −13.74 ± 1.73 to −4.91 ± 0.75 mV by the addition of IGF-1 and back to −13.52 ± 3.9 mV by the additional application of La3+), indicating the activation of a nonspecific cation conductance. The IGF-1–stimulated membrane conductance led to increases in intracellular free Ca2+. IGF-1 (50 ng/mL) increased cytosolic free Ca2+ from the base value to 1037 ± 111 nM, and the level was reduced by ruthenium red (20 μM) to 329 ± 22 nM (n = 11; P < 0.0001; Figs. 6E, 6F). IGF-1–dependent stimulation of TRPV2 channels and subsequent increases in intracellular free Ca2+ are known to be dependent on the activation of PI3-kinase. 25,32 Thus, we tested whether the IGF-1–induced Ca2+ increases were also dependent on PI3-kinase in RPE cells. In the presence of the PI3-kinase blocker LY294002 (30 μM) IGF-1 failed to increase cytosolic free Ca2+ (Fig. 6G). 
Figure 6.
 
IGF-1 induced membrane currents and increases in cytosolic free Ca2+. (A) Whole-cell currents at +60 mV during repeated stimulation with ramped voltages between −140 and +60 mV of 500-ms duration. The ramps were repeated every 5 seconds (holding potential −40 mV). IGF-1 was applied as indicated. Approximately 590 seconds after addition of IGF-1, a large whole-cell current appeared that was completely blocked by the additional application of 100 μM La3+. The numbers indicate the time points at which the representative ramps shown in (C) and (D) were taken. (B) Mock application of solution without IGF-1 had no effect on whole-cell currents. (C) Representative ramps of a cell stimulated by the application of IGF-1. (D) The same ramps as in (C) at higher magnification showing that currents induced by IGF-1 were completely abolished by the additional application of La3+. Note the shift of the reversal potential by IGF-1 induction and the shift back to control by La3+. (E) Ca2+ imaging with ARPE-19 cells: application of 50 ng/mL IGF-1 (bar) led to an increase in cytosolic free Ca2+ which was reduced during the additional presence of ruthenium red (20 μM; bar). (F) Summary of experiments of IGF-1 application and ruthenium red effects (n = 11 each group). (G) Ca2+ imaging with ARPE-19 cells: Application of the PI3-kinase inhibitor LY294002 (30 μM; bar) slightly increased cytosolic free Ca2+. Application of IGF-1 (50 ng/mL; bar) did not further change [Ca2+]i. (***P < 0.0001).
Figure 6.
 
IGF-1 induced membrane currents and increases in cytosolic free Ca2+. (A) Whole-cell currents at +60 mV during repeated stimulation with ramped voltages between −140 and +60 mV of 500-ms duration. The ramps were repeated every 5 seconds (holding potential −40 mV). IGF-1 was applied as indicated. Approximately 590 seconds after addition of IGF-1, a large whole-cell current appeared that was completely blocked by the additional application of 100 μM La3+. The numbers indicate the time points at which the representative ramps shown in (C) and (D) were taken. (B) Mock application of solution without IGF-1 had no effect on whole-cell currents. (C) Representative ramps of a cell stimulated by the application of IGF-1. (D) The same ramps as in (C) at higher magnification showing that currents induced by IGF-1 were completely abolished by the additional application of La3+. Note the shift of the reversal potential by IGF-1 induction and the shift back to control by La3+. (E) Ca2+ imaging with ARPE-19 cells: application of 50 ng/mL IGF-1 (bar) led to an increase in cytosolic free Ca2+ which was reduced during the additional presence of ruthenium red (20 μM; bar). (F) Summary of experiments of IGF-1 application and ruthenium red effects (n = 11 each group). (G) Ca2+ imaging with ARPE-19 cells: Application of the PI3-kinase inhibitor LY294002 (30 μM; bar) slightly increased cytosolic free Ca2+. Application of IGF-1 (50 ng/mL; bar) did not further change [Ca2+]i. (***P < 0.0001).
Discussion
We have identified, for the first time, the expression of TRPV channels in the RPE. In RPE cells, IGF-1 or heat induced enhanced ion conductance and VEGF-A secretion via TRPV channels. Analysis of transients of intracellular Ca2+ and membrane conductance revealed that TRPV2 channels most likely contributed to this activity. 
Many of the roles that the RPE plays in maintaining normal photoreceptor function are known to be regulated by changes in [Ca2+]i. 27,33 These roles include transcellular transport of amino acids, syntheses of inflammatory mediators, secretion of cytokines, and phagocytosis of shed photoreceptor outer segments. Nevertheless, the Ca2+ sources that provide changes in the [Ca2+]i level to mediate the regulation of these different functions are poorly understood. Freshly isolated hRPE cells expressed TRPV1, -2, -3, and -4 channels, as did the ARPE-19 cells. Stimulation by IGF-1 or increase in the temperature of RPE cells leads to increased VEGF-A secretion, which could be reduced by the TRPV channel blocker ruthenium red. Thus, we identified a new Ca2+-conductance in the RPE, carried by TRPV channels. 
To investigate the functional presence of TRPV channels in the RPE, we used a specific stimulus to activate these channels: an increase in temperature. 34 It is unlikely that these heat-evoked responses reflect a death signal of apoptosis after heat treatment. This notion is supported by the observations that the response could be repeatedly evoked; it was dependent on specific ion channels and did not reflect membrane damage; and in secretion experiments, the number of cells was not reduced 4 hours after heat treatment. Increasing the temperature in RPE cells led to an increase in intracellular free Ca2+ accompanied by an increase in an inwardly rectifying membrane conductance with a reversal potential of 0 mV. Considering the ion composition of the bath and pipette solutions, this reversal potential suggests the activity of either nonselective cation channels or Cl channels. Heat-induced Ca2+ increases were sensitive to La3+ and ruthenium red. This pharmacologic profile points to the activation of TRPV channels and therefore to activation of a nonselective cation conductance in the RPE. 34 The application of high concentrations of the different TRPV channel openers 2-APB, 30 camphor, 31 and capsaicin 30 showed that, of the four TRPV types, mainly TRPV2 was found to be active. Furthermore, that rather high temperatures were needed to evoke these effects leads to the conclusion that TRPV2 channels were active and involved in the heat-induced increases in intracellular Ca2+. This conclusion is supported by results obtained by using an siRNA approach to reduce the TRPV2 expression in ARPE-19 cells. After an efficient reduction of TRPV2 expression to 20% of the control expression, the heat-evoked increases in intracellular free Ca2+ were reduced to 27% of the normal Ca2+-transient. Last, IGF-1 led to an increase in intracellular free Ca2+ that was sensitive to ruthenium red. Thus, IGF-1–evoked Ca2+ transients were most likely due to activation of TRPV channels. 
In CHO cells, IGF-1 has been shown to increase the surface expression of heterologously expressed TRPV2 channels. 25 This TRPV2 current activation is mediated by the PI3-kinase. 25,32 Also, stimulation by IGF-1 led to an increase of inwardly rectifying membrane conductance that was blocked by La3+ in these cells. Thus, TRP channels can also be activated by IGF-1 in the RPE. 
The activation of these channels in the RPE led to enhanced secretion of VEGF-A. With the specific stimulus of these channels by an increase in temperature, ARPE-19 cells showed increased VEGF-A secretion in response to raised temperature. This increase in VEGF-A secretion was sensitive to ruthenium red, which confirms the possible activation of TPRV channels as mediators of this effect. The same was observed with IGF-1–stimulated VEGF-A secretion, which was also sensitive to the TRPV channel blocker ruthenium red. Thus, the involvement of TRPV implies two regulatory pathways for VEGF-A secretion by the RPE. One would be heat itself. The absorption of light by the RPE leads to an increase in the temperature of the RPE/choroid complex to values higher than 40°C. 35 In low concentrations, VEGF-A is fenestration factor for the endothelium of the choroid that seems to be involved in the temperature regulation of this complex. 35 Furthermore, this finding may explain the effects of laser treatment of the retina. 36 39 The other pathway would be the regulation of VEGF-A secretion by IGF-1. In previous works, we and other groups have shown that the stimulation of RPE cells with IGF-1 leads to the induction of VEGF secretion. 21 24 The intracellular mechanisms leading to increased VEGF secretion are only partly understood. Recently, it has been shown that IGF-1 influences the VEGF secretion by the RPE in two different pathways. Via activation of the phosphatidylinositol 3-kinase (PI3-kinase), IGF-1 leads to the stabilization of hypoxia-inducible factor (HIF)-1α, which in turn induces VEGF expression. 40 Although IGF-1–induced VEGF secretion is completely abolished by inhibition of PI3-kinase, the destabilization of HIF-1α only partly blocks VEGF secretion by the RPE, showing that there must be another HIF-1α-independent pathway. We found that IGF-1 simulated Ca2+ increase was inhibited by the PI3-kinase blocker LY294002. Thus, IGF-1–dependent stimulation of cytosolic free Ca2+ and subsequent VEGF-A secretion is promoted by activation of PI3-kinase and TRPV channels. 
As RPE-derived VEGF-A is known to play a major role in the induction of the formation of new blood vessels in age-related macular degeneration, these observations may help in understanding the underlying pathomechanisms. 5,7,10,41 Since IGF-1 plays a role in the induction of CNV, TRPV channels represent a target in the development of strategies to enhance the efficiency of anti-VEGF therapies. Furthermore, the heat-dependent stimulation of VEGF-A secretion by the RPE through activation of TRPV channels opens new insights into the understanding of laser treatments in patients or the CNV-causing effects in animal models of laser-induced CNV. 38,39,42 44  
In summary, we have shown that stimulation of the RPE by the addition of IGF-1 leads to a strong increase in VEGF secretion. This secretion is then probably mediated by the two pathways. One is the already-known pathway including the activation of PI3-kinase and subsequent stronger transcription of HIF-1α, as described herein. The other is the HIF-1α-independent enhancement of VEGF secretion. This pathway involves the PI3-kinase dependent activation of TRPV2 channels. With their major role in the VEGF secretion pathway in the RPE, TRPV2 channels may be a good target for VEGF-regulating therapy in retinal diseases with CNV as the major complication. 
Footnotes
 Supported by the Deutsche Forschungsgemeinschaft (DFG) Grants STR480/8-1 and 8-2.
Footnotes
 Disclosure: S. Cordeiro, None; S. Seyler, None; J. Stindl, None; V.M. Milenkovic, None; O. Strauss, None
The authors thank Elfriede Eckert for expert technical assistance and David Slattery for improving the language. 
References
Klein R Klein BE Linton KL . Prevalence of age-related maculopathy. The Beaver Dam Eye Study. Ophthalmology. 1992;99:933–943. [CrossRef] [PubMed]
Mitchell P Smith W Attebo K Wang JJ . Prevalence of age-related maculopathy in Australia: The Blue Mountains Eye Study. Ophthalmology. 1995;102:1450–1460. [CrossRef] [PubMed]
Vingerling JR Dielemans I Hofman A . The prevalence of age-related maculopathy in the Rotterdam Study. Ophthalmology. 1995;102:205–210. [CrossRef] [PubMed]
Vingerling JR Klaver CC Hofman A de Jong PT . Epidemiology of age-related maculopathy. Epidemiol Rev. 1995;17:347–360. [PubMed]
Bressler NM Bressler SB Fine SL . Age-related macular degeneration. Surv Ophthalmol. 1988;32:375–413. [CrossRef] [PubMed]
Votruba M Gregor Z . Neovascular age-related macular degeneration: present and future treatment options. Eye. 2001;15:424–429. [CrossRef] [PubMed]
Frank RN . Growth factors in age-related macular degeneration: pathogenic and therapeutic implications. Ophthalmic Res. 1997;29:341–353. [CrossRef] [PubMed]
Schmidt-Erfurth UM Pruente C . Management of neovascular age-related macular degeneration. Prog Retin Eye Res. 2007;26:437–451. [CrossRef] [PubMed]
Strauss O Boulton M . Choroidal neovascularisation: new dynamics of the VEGF signalling system. Exp Rev Ophthalmol. 2007;2:551–556. [CrossRef]
Amin R Puklin JE Frank RN . Growth factor localization in choroidal neovascular membranes of age-related macular degeneration. Invest Ophthalmol Vis Sci. 1994;35:3178–3188. [PubMed]
Dawson DW Volpert OV Gillis P . Pigment epithelium-derived factor: a potent inhibitor of angiogenesis. Science. 1999;285:245–248. [CrossRef] [PubMed]
Campochiaro PA . Retinal and choroidal neovascularization. J Cell Physiol. 2000;184:301–310. [CrossRef] [PubMed]
Kociok N Heppekausen H Schraermeyer U . The mRNA expression of cytokines and their receptors in cultured iris pigment epithelial cells: a comparison with retinal pigment epithelial cells. Exp Eye Res. 1998;67:237–250. [CrossRef] [PubMed]
Tanihara H Inatani M Honda Y . Growth factors and their receptors in the retina and pigment epithelium. Prog Retin Eye Res. 1997;16:271–301. [CrossRef]
Bian ZM Elner SG Elner VM . Thrombin-induced VEGF expression in human retinal pigment epithelial cells. Invest Ophthalmol Vis Sci. 2007;48:2738–2746. [CrossRef] [PubMed]
Ma W Lee SE Guo J . RAGE ligand upregulation of VEGF secretion in ARPE-19 cells. Invest Ophthalmol Vis Sci. 2007;48:1355–1361. [CrossRef] [PubMed]
Nagineni CN Samuel W Nagineni S . Transforming growth factor-beta induces expression of vascular endothelial growth factor in human retinal pigment epithelial cells: involvement of mitogen-activated protein kinases. J Cell Physiol. 2003;197:453–462. [CrossRef] [PubMed]
Punglia RS Lu M Hsu J . Regulation of vascular endothelial growth factor expression by insulin-like growth factor I. Diabetes. 1997;46:1619–1626. [CrossRef] [PubMed]
Rosenthal R Malek G Salomon N . The fibroblast growth factor receptors, FGFR-1 and FGFR-2, mediate two independent signalling pathways in human retinal pigment epithelial cells. Biochem Biophys Res Commun. 2005;337:241–247. [CrossRef] [PubMed]
Smith LE Kopchick JJ Chen W . Essential role of growth hormone in ischemia-induced retinal neovascularization. Science. 1997;276:1706–1709. [CrossRef] [PubMed]
Smith LE Shen W Perruzzi C . Regulation of vascular endothelial growth factor-dependent retinal neovascularization by insulin-like growth factor-1 receptor. Nat Med. 1999;5:1390–1395. [CrossRef] [PubMed]
Rosenthal R Wohlleben H Malek G . Insulin-like growth factor-1 contributes to neovascularization in age-related macular degeneration. Biochem Biophys Res Commun. 2004;323:1203–1208. [CrossRef] [PubMed]
Slomiany MG Rosenzweig SA . IGF-1-induced VEGF and IGFBP-3 secretion correlates with increased HIF-1 alpha expression and activity in retinal pigment epithelial cell line D407. Invest Ophthalmol Vis Sci. 2004;45:2838–2847. [CrossRef] [PubMed]
Slomiany MG Rosenzweig SA . Autocrine effects of IGF-I-induced VEGF and IGFBP-3 secretion in retinal pigment epithelial cell line ARPE-19. Am J Physiol Cell Physiol. 2004;287:C746–C753. [CrossRef] [PubMed]
Kanzaki M Zhang YQ Mashima H Li L Shibata H Kojima I . Translocation of a calcium-permeable cation channel induced by insulin-like growth factor-I. Nat Cell Biol. 1999;1:165–170. [CrossRef] [PubMed]
Van Buren JJ Bhat S Rotello R Pauza ME Premkumar LS . Sensitization and translocation of TRPV1 by insulin and IGF-I. Mol Pain. 2005;1:17. [CrossRef] [PubMed]
Wimmers S Karl MO Strauss O . Ion channels in the RPE. Prog Retin Eye Res. 2007;26:263–301. [CrossRef] [PubMed]
Vandesompele J De Preter K Pattyn F . Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002;3:RESEARCH0034. [CrossRef] [PubMed]
Grynkiewicz G Poenie M Tsien RY . A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985;260:3440–3450. [PubMed]
Vriens J Appendino G Nilius B . Pharmacology of vanilloid transient receptor potential cation channels. Mol Pharmacol. 2009;75:1262–1279. [CrossRef] [PubMed]
Sherkheli MA Benecke H Doerner JF . Monoterpenoids induce agonist-specific desensitization of transient receptor potential vanilloid-3 (TRPV3) ion channels. J Pharm Pharm Sci. 2009;12:116–128. [PubMed]
Boels K Glassmeier G Herrmann D . The neuropeptide head activator induces activation and translocation of the growth-factor-regulated Ca(2+)-permeable channel GRC. J Cell Sci. 2001;114:3599–3606. [PubMed]
Strauss O . The retinal pigment epithelium in visual function. Physiol Rev. 2005;85:845–881. [CrossRef] [PubMed]
Lee H Caterina MJ . TRPV channels as thermosensory receptors in epithelial cells. Pflugers Arch. 2005;451:160–167. [CrossRef] [PubMed]
Parver LM Auker C Carpenter DO . Choroidal blood flow as a heat dissipating mechanism in the macula. Am J Ophthalmol. 1980;89:641–646. [CrossRef] [PubMed]
Flaxel C Bradle J Acott T Samples JR . Retinal pigment epithelium produces matrix metalloproteinases after laser treatment. Retina. 2007;27:629–634. [CrossRef] [PubMed]
Hattenbach LO Beck KF Pfeilschifter J Koch F Ohrloff C Schacke W . Pigment-epithelium-derived factor is upregulated in photocoagulated human retinal pigment epithelial cells. Ophthalmic Res. 2005;37:341–346. [CrossRef] [PubMed]
Ibarra MS Hsu J Mirza N . Retinal temperature increase during transpupillary thermotherapy: effects of pigmentation, subretinal blood, and choroidal blood flow. Invest Ophthalmol Vis Sci. 2004;45:3678–3682. [CrossRef] [PubMed]
Ogata N Ando A Uyama M Matsumura M . Expression of cytokines and transcription factors in photocoagulated human retinal pigment epithelial cells. Graefes Arch Clin Exp Ophthalmol. 2001;239:87–95. [CrossRef] [PubMed]
Slomiany MG Rosenzweig SA . Hypoxia-inducible factor-1-dependent and -independent regulation of insulin-like growth factor-1-stimulated vascular endothelial growth factor secretion. J Pharmacol Exp Ther. 2006;318:666–675. [CrossRef] [PubMed]
Ishibashi T Hata Y Yoshikawa H Nakagawa K Sueishi K Inomata H . Expression of vascular endothelial growth factor in experimental choroidal neovascularization. Graefes Arch Clin Exp Ophthalmol. 1997;235:159–167. [CrossRef] [PubMed]
Ishida K Yoshimura N Yoshida M Honda Y . Upregulation of transforming growth factor-beta after panretinal photocoagulation. Invest Ophthalmol Vis Sci. 1998;39:801–807. [PubMed]
Morimura Y Okada AA Hayashi A . Histological effect and protein expression in subthreshold transpupillary thermotherapy in rabbit eyes. Arch Ophthalmol. 2004;122:1510–1515. [CrossRef] [PubMed]
Xiao M McLeod D Cranley J Williams G Boulton M . Growth factor staining patterns in the pig retina following retinal laser photocoagulation. Br J Ophthalmol. 1999;83:728–736. [CrossRef] [PubMed]
Figure 1.
 
Expression of TRPV channels in the human RPE and VEGF secretion by RPE cells. (A) Left: expression profile of TRPV channels in freshly isolated hRPE cells. Right: expression profile of TRPV channels in ARPE-19 cells. (B) IGF-1 induced secretion of VEGF-A by ARPE-19 cells. Shown is the normalized VEGF concentration in the medium. After stimulation by IGF-1 (50 ng/mL) and an additional 4-hour incubation at 37°C, the VEGF concentration in the medium was determined by ELISA. The values were normalized to control measurements without heat stimulus. In the presence of ruthenium red (20 μM), the IGF-1–induced VEGF secretion is decreased (n = 3). (C) Heat-induced secretion of VEGF-A by ARPE-19 cells. The normalized VEGF concentration in the medium is shown. Then, after a 5-minute heat-stimulus to 56°C and an additional 30-minute incubation at 37°C, the VEGF concentration in the medium was determined by ELISA. The values were normalized to control measurements without heat stimulus. The heat stimulation in the presence of ruthenium red (20 μM) clearly suppressed the increased VEGF secretion (n = 3). (*P < 0.05).
Figure 1.
 
Expression of TRPV channels in the human RPE and VEGF secretion by RPE cells. (A) Left: expression profile of TRPV channels in freshly isolated hRPE cells. Right: expression profile of TRPV channels in ARPE-19 cells. (B) IGF-1 induced secretion of VEGF-A by ARPE-19 cells. Shown is the normalized VEGF concentration in the medium. After stimulation by IGF-1 (50 ng/mL) and an additional 4-hour incubation at 37°C, the VEGF concentration in the medium was determined by ELISA. The values were normalized to control measurements without heat stimulus. In the presence of ruthenium red (20 μM), the IGF-1–induced VEGF secretion is decreased (n = 3). (C) Heat-induced secretion of VEGF-A by ARPE-19 cells. The normalized VEGF concentration in the medium is shown. Then, after a 5-minute heat-stimulus to 56°C and an additional 30-minute incubation at 37°C, the VEGF concentration in the medium was determined by ELISA. The values were normalized to control measurements without heat stimulus. The heat stimulation in the presence of ruthenium red (20 μM) clearly suppressed the increased VEGF secretion (n = 3). (*P < 0.05).
Figure 2.
 
Heat evoked increases in [Ca2+]i. Increasing the bath temperature from room temperature to 56°C led to an increase in [Ca2+]i in (A) ARPE-19 and (B) hRPE cells. (C) Downregulation of TRPV2 expression in ARPE-19 cells by siRNA: treatment of RPE cells with siRNA against TRPV2 decreased TRPV2 expression compared with cells treated with scrambled siRNA (n = 3 each group). (D) Heat-induced Ca2+ transients in ARPE-19 cells treated with siRNA against TRPV2 (n = 12 scrambled siRNA; n = 14 siRNA; **P < 0.01; ***P < 0.0001).
Figure 2.
 
Heat evoked increases in [Ca2+]i. Increasing the bath temperature from room temperature to 56°C led to an increase in [Ca2+]i in (A) ARPE-19 and (B) hRPE cells. (C) Downregulation of TRPV2 expression in ARPE-19 cells by siRNA: treatment of RPE cells with siRNA against TRPV2 decreased TRPV2 expression compared with cells treated with scrambled siRNA (n = 3 each group). (D) Heat-induced Ca2+ transients in ARPE-19 cells treated with siRNA against TRPV2 (n = 12 scrambled siRNA; n = 14 siRNA; **P < 0.01; ***P < 0.0001).
Figure 3.
 
Effect of the TPRV channel blocker La3+ and ruthenium red in RPE cells. The effect of the blocker La3+ (100 μM) and ruthenium red (1 μM) were investigated by using two heat steps (increase from room temperature to 56°C) in succession: the first without blocker, and the second with blocker. Statistical tests were performed to compare the increase in Ca2+ with versus without blocker. Original recordings showing the effects of (A) La3+ (100 μM; bar) and (B) ruthenium red (1 μM; bar) on heat-evoked Ca2+ increases in ARPE-19 cells. Original recordings showing the effects of (C) La3+ (100 μM; bar) and (D) ruthenium red (1 μM; bar) on heat-evoked Ca2+ increases in hRPE cells in primary culture. Comparison of the second heat-evoked Ca2+ increase (as a percentage of the first one), without and with (E) La3+ and (F) ruthenium red in ARPE-19 cells as well as in hRPE cells in primary culture. In the presence of both La3+ and ruthenium red the heat-evoked Ca2+ increases were significantly reduced. (*P < 0.05).
Figure 3.
 
Effect of the TPRV channel blocker La3+ and ruthenium red in RPE cells. The effect of the blocker La3+ (100 μM) and ruthenium red (1 μM) were investigated by using two heat steps (increase from room temperature to 56°C) in succession: the first without blocker, and the second with blocker. Statistical tests were performed to compare the increase in Ca2+ with versus without blocker. Original recordings showing the effects of (A) La3+ (100 μM; bar) and (B) ruthenium red (1 μM; bar) on heat-evoked Ca2+ increases in ARPE-19 cells. Original recordings showing the effects of (C) La3+ (100 μM; bar) and (D) ruthenium red (1 μM; bar) on heat-evoked Ca2+ increases in hRPE cells in primary culture. Comparison of the second heat-evoked Ca2+ increase (as a percentage of the first one), without and with (E) La3+ and (F) ruthenium red in ARPE-19 cells as well as in hRPE cells in primary culture. In the presence of both La3+ and ruthenium red the heat-evoked Ca2+ increases were significantly reduced. (*P < 0.05).
Figure 4.
 
Further pharmacologic analysis of the effect of TRPV channel openers on [Ca2+]i in ARPE-19 cells. (A) High concentrations of the TRPV2 channel opener 2-APB (3 mM; bar) led to an increase in [Ca2+]i. (B) The TRPV3 opener camphor (400 μM ; bar) did not change the level of [Ca2+]i. (C) The TRPV1 opener capsaicin (1 μM; bar) did not change the level of [Ca2+]i. (D) Relative increases in [Ca2+]i in ARPE-19 cells or in RPE cells in primary culture by 3 mM 2-APB (n = 3 each group).
Figure 4.
 
Further pharmacologic analysis of the effect of TRPV channel openers on [Ca2+]i in ARPE-19 cells. (A) High concentrations of the TRPV2 channel opener 2-APB (3 mM; bar) led to an increase in [Ca2+]i. (B) The TRPV3 opener camphor (400 μM ; bar) did not change the level of [Ca2+]i. (C) The TRPV1 opener capsaicin (1 μM; bar) did not change the level of [Ca2+]i. (D) Relative increases in [Ca2+]i in ARPE-19 cells or in RPE cells in primary culture by 3 mM 2-APB (n = 3 each group).
Figure 5.
 
Whole-cell recordings of heat-evoked membrane currents. (A) Changes in intracellular free Ca2+ by temperature increase to 45°C. After a recovery time of 25 minutes, the cells showed a full response to an increase to 56°C, as well. (B) Recording of the whole-cell recording of membrane currents. The membrane conductance potential was read by clamping to −40 mV, and the cell was electrically stimulated by voltage-ramps between −140 and +60 mV every 1.5 seconds. Left: recording showing the effect of increasing the bath temperature from room temperature to +45°C (arrow). Shortly after the temperature was increased, the membrane conductance increased. First, a transient inwardly rectifying conductance was observed that was followed by the sustained activation of a nonrectifying conductance, which remained sustained. Middle: recording of the whole-cell currents at −40 mV holding potential showing the fast transient heat-evoked (arrow) inward current. Right: comparison of whole-cell currents elicited by ramped voltages before and during an increase in the temperature to 45°C. (C) Recording of the whole-cell membrane currents in the presence of ruthenium red (20 μM). To monitor the membrane conductance the membrane potential was clamped to −40 mV and the cell was electrically stimulated by ramped voltages between −140 and +60 mV with increases every 1.5 seconds. Left: the effect of increasing the bath temperature from room temperature to +45°C (arrow). In the presence of ruthenium red, increasing the temperature failed to induce the transient inward current. Middle: recording of the whole-cell currents only at −40 mV holding potential in the presence of ruthenium red showing that increasing the temperature to 45°C failed to activate the transient inward current. Right: comparison of whole-cell currents elicited by ramped voltages, before and during an increase from room temperature to 45°C in the presence of ruthenium red.
Figure 5.
 
Whole-cell recordings of heat-evoked membrane currents. (A) Changes in intracellular free Ca2+ by temperature increase to 45°C. After a recovery time of 25 minutes, the cells showed a full response to an increase to 56°C, as well. (B) Recording of the whole-cell recording of membrane currents. The membrane conductance potential was read by clamping to −40 mV, and the cell was electrically stimulated by voltage-ramps between −140 and +60 mV every 1.5 seconds. Left: recording showing the effect of increasing the bath temperature from room temperature to +45°C (arrow). Shortly after the temperature was increased, the membrane conductance increased. First, a transient inwardly rectifying conductance was observed that was followed by the sustained activation of a nonrectifying conductance, which remained sustained. Middle: recording of the whole-cell currents at −40 mV holding potential showing the fast transient heat-evoked (arrow) inward current. Right: comparison of whole-cell currents elicited by ramped voltages before and during an increase in the temperature to 45°C. (C) Recording of the whole-cell membrane currents in the presence of ruthenium red (20 μM). To monitor the membrane conductance the membrane potential was clamped to −40 mV and the cell was electrically stimulated by ramped voltages between −140 and +60 mV with increases every 1.5 seconds. Left: the effect of increasing the bath temperature from room temperature to +45°C (arrow). In the presence of ruthenium red, increasing the temperature failed to induce the transient inward current. Middle: recording of the whole-cell currents only at −40 mV holding potential in the presence of ruthenium red showing that increasing the temperature to 45°C failed to activate the transient inward current. Right: comparison of whole-cell currents elicited by ramped voltages, before and during an increase from room temperature to 45°C in the presence of ruthenium red.
Figure 6.
 
IGF-1 induced membrane currents and increases in cytosolic free Ca2+. (A) Whole-cell currents at +60 mV during repeated stimulation with ramped voltages between −140 and +60 mV of 500-ms duration. The ramps were repeated every 5 seconds (holding potential −40 mV). IGF-1 was applied as indicated. Approximately 590 seconds after addition of IGF-1, a large whole-cell current appeared that was completely blocked by the additional application of 100 μM La3+. The numbers indicate the time points at which the representative ramps shown in (C) and (D) were taken. (B) Mock application of solution without IGF-1 had no effect on whole-cell currents. (C) Representative ramps of a cell stimulated by the application of IGF-1. (D) The same ramps as in (C) at higher magnification showing that currents induced by IGF-1 were completely abolished by the additional application of La3+. Note the shift of the reversal potential by IGF-1 induction and the shift back to control by La3+. (E) Ca2+ imaging with ARPE-19 cells: application of 50 ng/mL IGF-1 (bar) led to an increase in cytosolic free Ca2+ which was reduced during the additional presence of ruthenium red (20 μM; bar). (F) Summary of experiments of IGF-1 application and ruthenium red effects (n = 11 each group). (G) Ca2+ imaging with ARPE-19 cells: Application of the PI3-kinase inhibitor LY294002 (30 μM; bar) slightly increased cytosolic free Ca2+. Application of IGF-1 (50 ng/mL; bar) did not further change [Ca2+]i. (***P < 0.0001).
Figure 6.
 
IGF-1 induced membrane currents and increases in cytosolic free Ca2+. (A) Whole-cell currents at +60 mV during repeated stimulation with ramped voltages between −140 and +60 mV of 500-ms duration. The ramps were repeated every 5 seconds (holding potential −40 mV). IGF-1 was applied as indicated. Approximately 590 seconds after addition of IGF-1, a large whole-cell current appeared that was completely blocked by the additional application of 100 μM La3+. The numbers indicate the time points at which the representative ramps shown in (C) and (D) were taken. (B) Mock application of solution without IGF-1 had no effect on whole-cell currents. (C) Representative ramps of a cell stimulated by the application of IGF-1. (D) The same ramps as in (C) at higher magnification showing that currents induced by IGF-1 were completely abolished by the additional application of La3+. Note the shift of the reversal potential by IGF-1 induction and the shift back to control by La3+. (E) Ca2+ imaging with ARPE-19 cells: application of 50 ng/mL IGF-1 (bar) led to an increase in cytosolic free Ca2+ which was reduced during the additional presence of ruthenium red (20 μM; bar). (F) Summary of experiments of IGF-1 application and ruthenium red effects (n = 11 each group). (G) Ca2+ imaging with ARPE-19 cells: Application of the PI3-kinase inhibitor LY294002 (30 μM; bar) slightly increased cytosolic free Ca2+. Application of IGF-1 (50 ng/mL; bar) did not further change [Ca2+]i. (***P < 0.0001).
Table 1.
 
Oligonucleotides Used for the Detection of TRPV Channel Subunits
Table 1.
 
Oligonucleotides Used for the Detection of TRPV Channel Subunits
Protein (Accession No.*) Oligonucleotide Sequences Annealing Temp. Degrees (bp)
TRPV1 (NM_080704) Forward 5′-GCCTGGAGCTGTTCAAGTTC-3′ 59 (177)
Reverse 5′-TCTCCTGTGCGATCTTGTTG-3′
TRPV2 (NM_016113) Forward 5′-CAAACCGATTTGACCGAGAT-3′ 59 (167)
Reverse 5′-GTTCAGCACAGCCTTCATCA-3′
TRPV3 (NM_145068) Forward 5′-ACGAGGCAACAACATCCTTC-3′ 56 (226)
Reverse 5′-CCGCTTCTCCTTGATCTCAC-3′
TRPV4 (NM_147204) Forward 5′-GACGGGGACCTATAGCATCA-3′ 59 (228)
Reverse 5′-AACAGGTCCAGGAGGAAGGT-3′
TRPV5 (NM_019841) Forward 5′-GGAGCTTGTGGTCTCCTCTG-3′ 59 (185)
Reverse 5′-GAAACTTAAGGGGGCGGTAG-3′
TRPV6 (NM_018646) Forward 5′-GCCTATGGAGCAAGTTCTGC-3′ 50 (242)
Reverse 5′-GGCCTCCAGGTTGTCATAGA-3′
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×