Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta University, Augusta, GA, United States of America.
PLoS One. 2024 Apr 17;19(4):e0301495. doi: 10.1371/journal.pone.0301495. eCollection 2024.
The purpose of this study was to examine transient plasma membrane disruptions (TPMDs) and TPMD-induced Ca++ waves (TPMD Ca++ Wvs) in human and mouse corneal epithelium (HCEC and MCEC). A multi-photon microscope was used to create laser-induced TPMDs in single cultured cells and in intact ex vivo and in vivo MCECs and ex vivo human cornea rim HCECs. Eye rubbing-induced TPMDs were studied by gentle rubbing with a cotton tipped applicator over a closed eyelid in ex vivo and in vivo MCECs. Ca++ sources for TPMD-induced Ca++ waves were explored using Ca++ channel inhibitors and Ca++-free media. TPMDs and TPMD Ca++ Wvs were observed in all cornea epithelial models examined, often times showing oscillating Ca++ levels. The sarcoplasmic reticulum Ca++ ATPase inhibitors thapsigargin and CPA reduced TPMD Ca++ Wvs. TRP V1 antagonists reduced TPMD Ca++ Wvs in MCECs but not HCECs. Ca++-free medium, 18α-GA (gap junction inhibitor), apyrase (hydrolyzes ATP), and AMTB (TRPM8 inhibitor) did not affect TPMD Ca++ Wvs. These results provide a direct demonstration of corneal epithelial cell TPMDs and TPMDs in in vivo cells from a live animal. TPMDs were observed following gentle eye rubbing, a routine corneal epithelial cell mechanical stress, indicating TPMDs and TPMD Ca++ Wvs are common features in corneal epithelial cells that likely play a role in corneal homeostasis and possibly pathophysiological conditions. Intracellular Ca++ stores are the primary Ca++ source for corneal epithelial cell TPMD Ca++ Wvs, with TRPV1 Ca++ channels providing Ca++ in MCECs but not HCECs. Corneal epithelial cell TPMD Ca++ Wv propagation is not influenced by gap junctions or ATP.
本研究旨在检测人及鼠角膜上皮细胞(transient plasma membrane disruptions，TPMDs) 及 TPMD 诱导的钙波（transient plasma membrane disruptions Ca++ waves，TPMD Ca++ Wvs）。我们使用多光子显微镜在单细胞培养物及完整的离体和在体鼠角膜上皮细胞（mouse corneal epithelium，MCEC）和离体人角膜缘上皮细胞（human corneal epithelium，HCEC）中激光诱导 TPMD。通过在离体和在体 MCEC 及闭眼时用棉花棒轻柔摩擦，研究了眼摩擦诱导的 TPMD。我们通过使用 Ca++通道抑制剂和无 Ca++介质来研究 TPMD 诱导的 Ca++波的 Ca++来源。我们在所有检查的角膜上皮模型中均观察到 TPMDs 和 TPMD Ca++ Wvs，且这些模型常显示出波动的 Ca++水平。内质网 Ca++-ATP 酶抑制剂 thapsigargin 和 CPA 减少了 TPMD Ca++ Wvs。TRPV1 拮抗剂减少了 MCEC 中的 TPMD Ca++ Wvs，但对 HCEC 无影响。无 Ca++介质、18α-GA（缝隙连接抑制剂）、apyrase（水解 ATP）和 AMTB（TRPM8 抑制剂）不影响 TPMD Ca++ Wvs。这些结果直接证明了活体动物角膜上皮细胞中的 TPMDs 和 TPMD Ca++ Wvs。轻柔眼摩擦后观察到 TPMDs，这是一种常规的角膜上皮细胞机械应激，表明 TPMDs 和 TPMD Ca++ Wvs 是角膜上皮细胞的共同特征，可能在角膜稳态和可能的病理生理条件中发挥作用。细胞内 Ca++库是角膜上皮细胞 TPMD Ca++ Wvs 的主要 Ca++来源，TRPV1 Ca++ 通道在 MCEC 中提供 Ca++，但在 HCEC 中无作用。角膜上皮细胞 TPMD Ca++ Wv 的传播不受缝隙连接或 ATP 的影响。
