Department of Biology, University of North Florida, Jacksonville, FL, 32224, USA.
, Jacksonville, USA.
J Comp Physiol B. 2019 Oct;189(5):513-521. doi: 10.1007/s00360-019-01227-7. Epub 2019 Jul 17.
Previous work with isolated outer membrane vesicles of lobster branchiostegite epithelial cells has shown that Ca uptake by these structures is significantly (p < 0.02) reduced by an incremental decrease in saline pH (increased proton concentration) and that this decrease is due to competitive inhibition between carrier-mediated transport of Ca and hydrogen ions. The present paper extends these previous findings and describes the combined effects of pH and cationic heavy metals on branchiostegite uptake of Ca. Partially purified membrane vesicles of branchiostegite cells were produced by a homogenization/centrifugation method and were loaded with mannitol at pH 7.0. The time course of 1 mM Ca uptake in a mannitol medium at pH 8.5 containing 100 µM verapamil (Ca channel blocker) was hyperbolic and approached equilibrium at 30 min. This uptake was either significantly reduced (p < 0.05) by the addition of 5 µM Zn or essentially abolished with the addition of 5 µM Cu. Increasing zinc concentrations (5-500 µM) reduced 1 mM Ca uptake at pH 8.5 or 7.5 in a hyperbolic fashion with the remaining non-inhibited uptake due to apparent non-specific binding. Uptake of 1 mM Ca at pH 8.5, 7.5, 7.5 + Zn, and 7.5 + Zn + Cu + Cd in the presence of 100 µM verapamil displayed a stepwise reduction of Ca uptake with the addition of each treatment until only non-specific isotope binding occurred with all cation inhibitors. Ca influxes (15 s uptakes; 0.25-5.0 mM calcium + 100 µM verapamil) in the presence and absence of 10 µM Zn were both hyperbolic functions of calcium concentration. The curve with Zn displayed a transport K twice that of the control (p < 0.05), while inhibitor and control curve J values were not significantly different (p > 0.05), suggesting competitive inhibition between Ca and Zn influxes. Analysis of the relative inhibitory effects of increased proton or heavy metal interaction with Ca uptake suggests that divalent metals may reduce the calcium transport about twice as much as a drop in pH, but together, they appear to abolish carrier-mediated transport.
先前对龙虾鳃足上皮细胞的分离的外膜囊泡的研究表明，这些结构对 Ca 的摄取会随着盐溶液 pH 值（质子浓度增加）的逐渐降低而显著减少（p<0.02），这种减少是由于载体介导的 Ca 和氢离子的运输之间的竞争性抑制。本论文扩展了这些先前的发现，并描述了 pH 值和阳离子重金属对鳃足吸收 Ca 的综合影响。通过匀浆/离心法制备鳃足细胞的部分纯化膜囊泡，并在 pH 7.0 下用甘露醇装载。在含有 100 µM 维拉帕米（钙通道阻滞剂）的甘露醇介质中，1 mM Ca 的摄取时间过程呈双曲线，在 30 分钟时达到平衡。添加 5 µM Zn 可显著降低（p<0.05）这种摄取，而添加 5 µM Cu 则基本消除了这种摄取。随着锌浓度（5-500 µM）的增加，在 pH 8.5 或 7.5 下，1 mM Ca 的摄取以双曲线方式减少，而剩余的非抑制性摄取归因于明显的非特异性结合。在 100 µM 维拉帕米存在下，在 pH 8.5、7.5、7.5+Zn 和 7.5+Zn+Cu+Cd 下 1 mM Ca 的摄取显示出 Ca 摄取的逐步减少，随着每种处理的添加，直到所有阳离子抑制剂仅发生非特异性同位素结合。在存在和不存在 10 µM Zn 的情况下，15 s 摄取（0.25-5.0 mM 钙+100 µM 维拉帕米）的 Ca 内流都是钙浓度的双曲线函数。Zn 曲线的转运 K 是对照的两倍（p<0.05），而抑制剂和对照曲线的 J 值没有显著差异（p>0.05），这表明 Ca 和 Zn 内流之间存在竞争性抑制。分析质子或重金属与 Ca 摄取增加的相互作用的相对抑制作用表明，二价金属可能会使 Ca 转运减少约两倍，而 pH 值降低则会减少一半，但它们似乎共同消除了载体介导的转运。
