Laboratory of Veterinary Physiology, Joint Department of Veterinary Medicine, Faculty of Agriculture, Tottori University, Tottori, Japan; The United Graduate School of Veterinary Science, Yamaguchi University, Yamaguchi, Japan.
Laboratory of Veterinary Physiology, Joint Department of Veterinary Medicine, Faculty of Agriculture, Tottori University, Tottori, Japan.
Cell Calcium. 2019 May;79:11-19. doi: 10.1016/j.ceca.2019.02.001. Epub 2019 Feb 6.
Rat melanotrophs express several types of voltage-gated and ligand-gated calcium channels, although mechanisms involved in the maintenance of the resting intracellular Ca concentration ([Ca]) remain unknown. We analyzed mechanisms regulating resting [Ca] in dissociated rat melanotrophs by Ca-imaging and patch-clamp techniques. Treatment with antagonists of L-type, but not N- or P/Q-type voltage-gated Ca channels (VGCCs) as well as removal of extracellular Ca resulted in a rapid and reversible decrease in [Ca], indicating constitutive Ca influx through L-type VGCCs. Reduction of extracellular Na concentration (replacement with NMDG) similarly decreased resting [Ca]. When cells were champed at -80 mV, decrease in the extracellular Na resulted in a positive shift of the holding current. In cell-attached voltage-clamp and whole-cell current-clamp configurations, the reduction of extracellular Na caused hyperpolarisation. The holding current shifted in negative direction when extracellular K concentration was increased from 5 mM to 50 mM in the presence of K channel blockers, Ba and TEA, indicating cation nature of persistent conductance. RT-PCR analyses of pars intermedia tissues detected mRNAs of TRPV1, TRPV4, TRPC6, and TRPM3-5. The TRPV channel blocker, ruthenium red, shifted the holding current in positive direction, and significantly decreased the resting [Ca]. These results indicate operation of a constitutive cation conductance sensitive to ruthenium red, which regulates resting membrane potential and [Ca] in rat melanotrophs.
大鼠黑色素细胞表达多种类型的电压门控和配体门控钙通道，尽管维持细胞内静息 Ca 浓度 ([Ca]) 的机制仍不清楚。我们通过钙成像和膜片钳技术分析了分离的大鼠黑色素细胞中调节静息 [Ca] 的机制。用 L 型电压门控钙通道 (VGCC) 而不是 N 型或 P/Q 型 VGCC 的拮抗剂处理以及去除细胞外 Ca 导致 [Ca] 快速和可逆下降，表明 L 型 VGCC 持续存在 Ca 内流。降低细胞外 Na 浓度（用 NMDG 取代）同样降低静息 [Ca]。当细胞在 -80 mV 钳位时，细胞外 Na 的减少导致保持电流的正移。在细胞贴附电压钳和全细胞电流钳配置中，降低细胞外 Na 导致超极化。当存在 K 通道阻滞剂 Ba 和 TEA 时，将细胞外 K 浓度从 5 mM 增加到 50 mM 会导致保持电流向负方向移动，表明持续电导具有阳离子性质。对中间部分组织进行的 RT-PCR 分析检测到 TRPV1、TRPV4、TRPC6 和 TRPM3-5 的 mRNA。TRPV 通道阻滞剂钌红使保持电流向正方向移动，并显著降低静息 [Ca]。这些结果表明，一种对钌红敏感的组成性阳离子电导起作用，调节大鼠黑色素细胞的静息膜电位和 [Ca]。
