Ocean acidification: synergistic inhibitory effects of protons and heavy metals on Ca uptake by lobster branchiostegite membrane vesicles.

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.