Department of Biology, University of North Florida, Jacksonville, FL, 32224, USA.
J Comp Physiol B. 2018 Sep;188(5):739-747. doi: 10.1007/s00360-018-1173-2. Epub 2018 Jul 14.
Gill chambers of the Atlantic lobster, Homarus americanus, possess three structures that are involved with respiration and ion regulation: gill filaments, epipodites, and branchiostegites. This paper describes ion transport mechanisms present in the plasma membranes of branchiostegite epithelial cells and the effects of pH on the uptake of Ca by these processes. Partially purified membrane vesicles (PPMV) of branchiostegite cells were produced by a homogenization/centrifugation method that has previously been used to define ion transport processes in both crab and lobster gill tissues. In the present study, lobster branchiostegite PPMV Ca uptake was highest at pH 8.5 and lowest at pH values between 6.0 and 7.0 (p < 0.02). At pH 8.0, Ca uptake was a biphasic process consisting of a saturable process at low [Ca] and a linear process at higher [Ca]. At pH 6.0, Ca uptake was only a linear process and paralleled linear uptake at pH 8.0. A valinomycin/K-induced membrane potential (PD, inside negative) doubled Ca uptake at pH 7.0 above that in the absence of a PD (p < 0.05). An induced PD at pH 8.0 did not significantly (p > 0.05) affect Ca uptake observed in the absence of a PD, but was threefold greater than uptake at pH 7.0 in the absence of a PD (p < 0.05). Amiloride (2 mM) did not affect Ca uptake at pH 8.0, but 2 mM amiloride + 100 µM verapamil reduced uptake by approximately 50%. In the presence of both 2 mM amiloride + 100 µM verapamil, 15 s Ca influx at pH 8.5 was a hyperbolic function of [Ca] (0.1-5 mM) (K = 4.2 ± 0.3 mM; J = 9792 ± 439 pmol/mg protein × 15 s). Ca influxes at pH 7.5 under the same conditions were also hyperbolic with K = 8.3 ± 1.4 mM; J = 10732 ± 1250 pmol/mg protein × 15 s. K values were significantly different (p < 0.05), but J values were not (p > 0.05). These results suggest that Ca uptake by lobster branchiostegites may have occurred by the combination of diffusion through a verapamil-inhibited calcium channel and carrier-mediated transport by amiloride-insensitive, electroneutral, 1Ca/2H antiporters. Decreased pH, as might occur during ocean acidification, did not appear to modify calcium diffusion through the channels, but protons acted as competitive inhibitors of calcium transport by carrier-mediated antiport. Decreased calcium uptake with continued ocean acidification may significantly affect calcification processes during periodic molting, potentially influencing mortality.
鳃丝、副肢和鳃弓。本文描述了存在于鳃弓上皮细胞质膜中的离子转运机制，以及 pH 值对这些过程中 Ca 摄取的影响。通过先前用于定义蟹类和龙虾鳃组织中离子转运过程的匀浆/离心方法，产生了部分纯化的鳃弓细胞质膜囊泡（PPMV）。在本研究中，龙虾鳃弓 PPMV 的 Ca 摄取在 pH8.5 时最高，在 pH6.0 和 7.0 之间时最低（p<0.02）。在 pH8.0 时，Ca 摄取是一个双相过程，包括在低[Ca]下的饱和过程和在高[Ca]下的线性过程。在 pH6.0 时，Ca 摄取仅是一个线性过程，与 pH8.0 时的线性摄取平行。缬氨霉素/K 诱导的膜电位（PD，内负）使 pH7.0 时的 Ca 摄取比无 PD 时增加一倍（p<0.05）。在无 PD 的情况下，诱导的 PD 对 pH8.0 时观察到的 Ca 摄取没有显著影响（p>0.05），但比无 PD 时 pH7.0 时的摄取高三倍（p<0.05）。阿米洛利（2 mM）对 pH8.0 时的 Ca 摄取没有影响，但 2 mM 阿米洛利+100 µM 维拉帕米可使摄取减少约 50%。在存在 2 mM 阿米洛利+100 µM 维拉帕米的情况下，pH8.5 时的 15 s Ca 内流是 [Ca]（0.1-5 mM）的双曲线函数（K = 4.2±0.3 mM；J = 9792±439 pmol/mg 蛋白×15 s）。在相同条件下，pH7.5 时的 Ca 内流也是双曲线的，K = 8.3±1.4 mM；J = 10732±1250 pmol/mg 蛋白×15 s。K 值有显著差异（p<0.05），但 J 值没有（p>0.05）。这些结果表明，龙虾鳃弓的 Ca 摄取可能是通过维拉帕米抑制的钙通道的扩散和阿米洛利不敏感的、电中性的、1Ca/2H 反向转运体介导的转运的组合来实现的。海洋酸化过程中可能发生的 pH 值降低似乎并没有改变钙通过通道的扩散，但质子作为载体介导的反向转运的 Ca 转运的竞争性抑制剂起作用。随着海洋酸化的持续进行，钙摄取的减少可能会显著影响周期性蜕皮过程中的钙化过程，从而可能影响死亡率。
