Calcium transport and homeostasis in gill cells of a freshwater crab Dilocarcinus pagei
Crustaceans present a very interesting model system to study the process of calcification and calcium (Ca2+) transport because of molting-related events and the deposition of CaCO3 in the new exoskeleton. Dilocarcinus pagei, a freshwater crab endemic to Brazil, was studied to understand Ca2+ transport in whole gill cells using a fluorescent probe. Cells were dissociated, all of the gill cell types were loaded with fluo-3 and intracellular Ca2+ change was monitored by adding Ca as CaCl2 (0, 0.1, 0.25, 0.50, 1.0 and 5 mM), with a series of different inhibitors. For control gill cells, Ca2+ transport followed Michaelis–Menten kinetics with Km = 0.42 ± 0.04 mM and Vmax = 0.50 ± 0.02 μM (Ca2+ change × initial intracellular Ca−1 × 180 s−1; N = 14, r2 = 0.99). Verapamil (a Ca2+ channel inhibitor) and amiloride (a Na+/Ca2+ exchanger [NCX] inhibitor) completely reduced intracellular Ca2+ transport, while nifedipine, another Ca2+ channel inhibitor, did not. Vanadate, a plasma membrane Ca2+-ATPase inhibitor (PMCA), increased intracellular Ca2+ in gill cells through a decrease in the efflux of Ca2+. Ouabain increased intracellular Ca2+, similar to the effect of KB-R, a specific NCX inhibitor for Ca2+ in the influx mode. Alterations in extracellular [Na] in the saline did not affect intracellular Ca2+ transport. Caffeine, responsible for inducing Ca release from sarcoplasmic reticulum in vertebrate muscle, increased intracellular Ca2+ compared to control, suggesting an effect of this inhibitor in gill epithelial cells of Dilocarcinus pagei, probably through release of intracellular stores. We also demonstrate here that intracellular Ca2+ in gill cells of Dilocarcinus pagei was kept relatively constant in face of an extracellular Ca concentration of 50-fold, suggesting that crustaceans are able to display Ca2+ homeostasis through various Ca2+ intracellular sequestration mechanisms and/or plasma membrane Ca2+ influx and outflux that are highly regulatory. In summary, studies using whole gill cells are an interesting approach for working with real regulatory Ca2+ mechanisms in intact cells under physiological Ca levels (mM range), compared to earlier work using isolated vesicles of various epithelial cells.