Previous work with isolated outer membrane vesicles of lobster branchiostegite epithelial cells has shown that 45Ca2+ 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 45Ca2+ 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 45Ca2+. 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 45Ca2+ uptake in a mannitol medium at pH 8.5 containing 100 µM verapamil (Ca2+ 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 Zn2+ or essentially abolished with the addition of 5 µM Cu2+. Increasing zinc concentrations (5–500 µM) reduced 1 mM 45Ca2+ 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 45Ca2+ at pH 8.5, 7.5, 7.5 + Zn2+, and 7.5 + Zn2+ + Cu2+ + Cd2+ in the presence of 100 µM verapamil displayed a stepwise reduction of 45Ca2+ uptake with the addition of each treatment until only non-specific isotope binding occurred with all cation inhibitors. 45Ca2+ influxes (15 s uptakes; 0.25–5.0 mM calcium + 100 µM verapamil) in the presence and absence of 10 µM Zn2+ were both hyperbolic functions of calcium concentration. The curve with Zn2+ displayed a transport Km twice that of the control (p < 0.05), while inhibitor and control curve Jmax values were not significantly different (p > 0.05), suggesting competitive inhibition between 45Ca2+ and Zn2+ influxes. Analysis of the relative inhibitory effects of increased proton or heavy metal interaction with 45Ca2+ 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.

中文翻译：

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海洋酸化：质子和重金属对龙虾枝条膜囊泡吸收 45Ca 的协同抑制作用

先前对龙虾鳃上皮细胞的分离外膜囊泡的研究表明，这些结构对 45Ca2+ 的吸收显着（p < 0.02）随着盐水 pH 值的逐渐降低（质子浓度增加）而降低，并且这种降低是由于之间的竞争性抑制。载体介导的 45Ca2+ 和氢离子转运。本论文扩展了这些先前的研究结果，并描述了 pH 值和阳离子重金属对 45Ca2+ 吸收枝晶石的综合影响。通过均质化/离心方法产生部分纯化的枝条细胞膜囊泡，并在 pH 7.0 下加载甘露醇。在 pH 8 的甘露醇培养基中吸收 1 mM 45Ca2+ 的时间过程。5 含有 100 µM 维拉帕米（Ca2+ 通道阻滞剂）呈双曲线并在 30 分钟时达到平衡。加入 5 µM Zn2+ 或加入 5 µM Cu2+ 后，这种吸收明显减少（p < 0.05）。增加锌浓度 (5–500 µM) 在 pH 8.5 或 7.5 下以双曲线方式减少 1 mM 45Ca2+ 的吸收，而由于明显的非特异性结合，剩余的非抑制吸收。在 100 µM 维拉帕米存在下，在 pH 8.5、7.5、7.5 + Zn2+ 和 7.5 + Zn2+ + Cu2+ + Cd2+ 条件下吸收 1 mM 45Ca2+ 显示出随着每次处理的添加逐步减少 45Ca2+ 吸收，直到只有非特异性同位素结合所有阳离子抑制剂均发生。45Ca2+ 流入（15 秒吸收；0.25–5. 0 mM 钙 + 100 µM 维拉帕米）在存在和不存在 10 µM Zn2+ 的情况下都是钙浓度的双曲线函数。Zn2+ 曲线的转运 Km 是对照的两倍（p < 0.05），而抑制剂和对照曲线的 Jmax 值没有显着差异（p > 0.05），表明 45Ca2+ 和 Zn2+ 流入之间存在竞争性抑制。对增加的质子或重金属相互作用与 45Ca2+ 摄取的相对抑制作用的分析表明，二价金属可能会降低钙转运的两倍，大约是 pH 下降的两倍，但它们加在一起似乎消除了载体介导的转运。表明 45Ca2+ 和 Zn2+ 流入之间的竞争性抑制。对增加的质子或重金属相互作用与 45Ca2+ 摄取的相对抑制作用的分析表明，二价金属可能会降低钙转运的两倍，大约是 pH 下降的两倍，但它们加在一起似乎消除了载体介导的转运。表明 45Ca2+ 和 Zn2+ 流入之间的竞争性抑制。对增加的质子或重金属相互作用与 45Ca2+ 摄取的相对抑制作用的分析表明，二价金属可能会降低钙转运的两倍，大约是 pH 下降的两倍，但它们加在一起似乎消除了载体介导的转运。