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) (K_m = 4.2 ± 0.3 mM; J_max = 9792 ± 439 pmol/mg protein × 15 s). ⁴⁵Ca influxes at pH 7.5 under the same conditions were also hyperbolic with K_m = 8.3 ± 1.4 mM; J_max = 10732 ± 1250 pmol/mg protein × 15 s. K_m values were significantly different (p < 0.05), but J_max 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.