Retinal pigment epithelial (RPE) cells express different subtypes of inwardly rectifying potassium (Kir) channels. We investigated whether human and rat RPE cells express genes of strongly rectifying Kir2 channels. We also determined the hypoxic and hyperosmotic regulation of Kir2.1 gene expression in cultured human RPE cells and the effects of siRNA-mediated knockdown of Kir2.1 on VEGFA expression, VEGF secretion, proliferation, and viability of the cells. Extracellular hyperosmolarity was induced by addition of NaCl or sucrose. Hypoxia and chemical hypoxia were produced by cell culture in 0.25% O₂ and addition of CoCl₂, respectively. Gene expression levels were evaluated by real-time RT-PCR. Rat RPE cells contained Kir2.1, Kir2.2, Kir2.3, and Kir2.4 gene transcripts while human RPE cells contained Kir2.1, Kir2.2, and Kir2.4 transcripts. Immunocytochemical data may suggest that Kir2.1 protein in cultured human cells is expressed in both perinuclear and plasma membranes. Kir2.1 gene expression and Kir2.1 protein level in human cells increased under hypoxic and hyperosmotic conditions. The expression of the Kir2.1 gene was mediated in part by diverse intracellular signal transduction pathways and transcription factor activities under both conditions; the hyperosmotic, but not the CoCl₂-induced Kir2.1 gene expression was dependent on intracellular calcium signaling. Autocrine/paracrine activation of purinergic receptors contributed to Kir2.1 gene expression under hyperosmotic (P2Y₁, P2Y₂, P2X₇) and CoCl₂-induced conditions (P2Y₂, P2X₇). Exogenous VEGF, TGF-β1, and blood serum decreased Kir2.1 gene expression. Inhibition of VEGF receptor-2 increased the Kir2.1 gene expression under control conditions and in CoCl₂-simulated hypoxia, and decreased it under high NaCl conditions. Knockdown of Kir2.1 by siRNA inhibited the CoCl₂-induced and hyperosmotic transcription of the VEGFA gene and caused a delayed decrease of the constitutive VEGFA gene expression while VEGF protein secretion was not altered. Kir2.1 knockdown stimulated RPE cell proliferation under control and hyperosmotic conditions without affecting cell viability. The data indicate that Kir2.1 channel activity is required for the expression of the VEGFA gene and inhibits the proliferation of RPE cells. Under control and hypoxic conditions, the extracellular VEGF level may regulate the production of VEGF via its inhibitory effect on the Kir2.1 gene transcription; this feedback loop may prevent overproduction of VEGF.

视网膜色素上皮 (RPE) 细胞表达不同亚型的内向整流钾 (Kir) 通道。我们研究了人和大鼠 RPE 细胞是否表达强烈矫正 Kir2 通道的基因。我们还确定了培养的人 RPE 细胞中Kir2.1基因表达的缺氧和高渗调节，以及 siRNA 介导的 Kir2.1 敲低对VEGFA表达、VEGF 分泌、增殖和细胞活力的影响。通过添加 NaCl 或蔗糖诱导细胞外高渗。缺氧和化学缺氧分别通过在 0.25% O ₂和添加 CoCl _{2 中}的细胞培养产生。通过实时RT-PCR评估基因表达水平。含有大鼠 RPE 细胞Kir2.1、Kir2.2、Kir2.3和Kir2.4基因转录本，而人类 RPE 细胞含有Kir2.1、Kir2.2和Kir2.4转录本。免疫细胞化学数据可能表明培养的人类细胞中的 Kir2.1 蛋白在核周膜和质膜中均有表达。在缺氧和高渗条件下，人体细胞中的Kir2.1基因表达和 Kir2.1 蛋白水平增加。在这两种条件下，Kir2.1基因的表达部分是由不同的细胞内信号转导途径和转录因子活性介导的；高渗，但不是 CoCl ₂诱导Kir2.1基因表达依赖于细胞内钙信号。嘌呤能受体的自分泌/旁分泌激活有助于在高渗（P2Y ₁、P2Y ₂、P2X ₇）和 CoCl ₂诱导条件（P2Y ₂、P2X ₇）下的Kir2.1基因表达。外源性 VEGF、TGF-β1 和血清降低了Kir2.1基因的表达。在对照条件和 CoCl ₂模拟缺氧条件下，VEGF 受体-2 的抑制增加了Kir2.1基因表达，并在高 NaCl 条件下降低了该基因表达。siRNA 对 Kir2.1 的敲低抑制了 CoCl ₂VEGFA基因的诱导和高渗转录，并导致组成型VEGFA基因表达的延迟降低，而 VEGF 蛋白分泌没有改变。Kir2.1 组合式在控制和高渗条件下刺激 RPE 细胞增殖，而不影响细胞活力。数据表明，Kir2.1 通道活性是VEGFA基因表达所必需的，并抑制 RPE 细胞的增殖。在控制和缺氧条件下，细胞外VEGF水平可能通过其对Kir2.1基因转录的抑制作用来调节VEGF的产生；这种反馈回路可以防止 VEGF 的过度产生。