Expression pattern of Kv11 (Ether à-go-go-related gene; erg) K+ channels in the mouse retina.

In response to light, most retinal neurons exhibit gradual changes in membrane potential. Therefore K+ channels that mediate threshold currents are well-suited for the fine-tuning of signal transduction. In the present study we demonstrate the expression of the different Kv11 (ether-à-go-go related gene; erg) channel subunits in the human and mouse retina by RT PCR and quantitative PCR, respectively. Immunofluorescence analysis with cryosections of mouse retinae revealed the following local distribution of the three Kv11 subunits: Kv11.1 (m-erg1) displayed the most abundant expression with the strongest immunoreactivity in rod bipolar cells. In addition, immunoreactivity was found in the inner part of the outer plexiform layer (OPL), in the inner plexiform layer (IPL) and in the inner segments of photoreceptors. Immunoreactivity for Kv11.2 (m-erg2) was observed in the outer part of the OPL and throughout the IPL. Double-labeling for vGluT1 or synaptophysin indicated a mainly presynaptic localization of Kv11.2. While no significant staining for Kv11.3 (m-erg3) was detected in the neuronal retina, strong Kv11.3 immunoreactivity was present in the apical membrane of the retinal pigment epithelium. The different expression levels were confirmed by real-time PCR showing almost equal levels of Kv11.1 and Kv11.2, while Kv11.3 mRNA expression was significantly lower. The two main splice variants of Kv11.1, isoforms a and b were detected in comparable levels suggesting a possible formation of cGMP/cGK-sensitive Kv11.1 channels in photoreceptors and rod bipolar cells. Taken together, the immunohistological results revealed different expression patterns of the three Kv11 channels in the mouse retina supposing distinct physiological roles.

Expression of Orai genes and I(CRAC) activation in the human retinal pigment epithelium.

BACKGROUND: The retinal pigment epithelium (RPE) fulfills a large variety of tasks that are important for visual function. Many of these tasks, such as phagocytosis, growth factor secretion, or transepithelial ion transport, are regulated by increases in intracellular Ca²(+) as second-messenger. Despite the multitude of Ca²(+)-dependently regulated functions, only few Ca²(+) channels have been described so far in the RPE to couple Ca²(+) conductance and Ca²(+) signaling. METHODS: RT-PCR experiments with mRNA of freshly isolated RPE cells as well as from the RPE cell line ARPE-19 and measurements of the intracellular free Ca²(+) concentration were performed. RESULTS: The RT-PCR experiments revealed the expression of the I(CRAC) channel proteins Orai 1, 2, and 3 and their stimulators Stim-1 and Stim-2. The classic maneuver to stimulate capacitive Ca²(+) entry (depletion of Ca²(+) stores by 1 µM thapsigargin under extracellular Ca²(+)-free conditions and then re-adding extracellular Ca²(+)) led to an increase in intracellular free Ca²(+), which could be blocked by application of a high concentration of 2-APB (75 µM) either before or during induction of capacitive Ca²(+) entry. On the other hand, application of a low concentration of 2-APB (2 µM) led to enhancement of the Ca²(+) increase induced by capacitive Ca²(+) entry. Depletion of cytosolic Ca²(+) stores by administration of an extracellular divalent cation-free solution led to an increase in the whole-cell conductance. CONCLUSIONS: With these data we show a new Ca²(+) entry pathway linked to the Ca²(+)/inositolphosphate second-messenger system in RPE cells which help to further understand regulatory pathways of agonists. The expression of Orai channels enables the RPE cells to generate sustained or repetitive Ca²(+) signals as they are known to be induced by different stimuli like ATP, bFGF, and the stimulation with photoreceptor outer segments.

Heat-sensitive TRPV channels in retinal pigment epithelial cells: regulation of VEGF-A secretion.

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: Ca(2+) signaling was studied in ARPE-19 and human RPE cells in primary culture by means of Ca(2+) 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 Ca(2+) signals were blocked by the TRPV channel blockers La(3+) and ruthenium red by 68% and 52%, respectively. In contrast, high concentrations of 2-APB (3 mM) increased [Ca(2+)](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 Ca(2+) 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 [Ca(2+)](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 Ca(2+) signaling that mediates both heat-dependent and IGF-1 (via PI3-kinase activation)-induced VEGF secretion.