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

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.

Ocean acidification: effects of pH on 45Ca uptake by lobster branchiostegites.

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 45Ca 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 45Ca 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, 45Ca uptake was a biphasic process consisting of a saturable process at low [Ca] and a linear process at higher [Ca]. At pH 6.0, 45Ca uptake was only a linear process and paralleled linear uptake at pH 8.0. A valinomycin/K+-induced membrane potential (PD, inside negative) doubled 45Ca 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 45Ca 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 45Ca 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 45Ca influx at pH 8.5 was a hyperbolic function of [Ca] (0.1-5 mM) (Km = 4.2 ± 0.3 mM; Jmax = 9792 ± 439 pmol/mg protein × 15 s). 45Ca influxes at pH 7.5 under the same conditions were also hyperbolic with Km = 8.3 ± 1.4 mM; Jmax = 10732 ± 1250 pmol/mg protein × 15 s. Km values were significantly different (p < 0.05), but Jmax values were not (p > 0.05). These results suggest that 45Ca 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, 1Ca2+/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.