Ocean acidification: synergistic inhibitory effects of protons and heavy metals on ⁴⁵Ca uptake by lobster branchiostegite membrane vesicles

Abstract

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 K_m twice that of the control (p < 0.05), while inhibitor and control curve J_max 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.