Calcium transport and homeostasis in gill cells of a freshwater crab Dilocarcinus pagei

Abstract

Crustaceans present a very interesting model system to study the process of calcification and calcium (Ca(2+)) transport because of molting-related events and the deposition of CaCO(3) in the new exoskeleton. Dilocarcinus pagei, a freshwater crab endemic to Brazil, was studied to understand Ca(2+) 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 Ca(2+) change was monitored by adding Ca as CaCl(2) (0, 0.1, 0.25, 0.50, 1.0 and 5 mM), with a series of different inhibitors. For control gill cells, Ca(2+) transport followed Michaelis-Menten kinetics with K(m) = 0.42 +/- 0.04 mM and V(max) = 0.50 +/- 0.02 microM (Ca(2+) change x initial intracellular Ca(-1) x 180 s(-1); N = 14, r (2) = 0.99). Verapamil (a Ca(2+) channel inhibitor) and amiloride (a Na(+)/Ca(2+) exchanger [NCX] inhibitor) completely reduced intracellular Ca(2+) transport, while nifedipine, another Ca(2+) channel inhibitor, did not. Vanadate, a plasma membrane Ca(2+)-ATPase inhibitor (PMCA), increased intracellular Ca(2+) in gill cells through a decrease in the efflux of Ca(2+). Ouabain increased intracellular Ca(2+), similar to the effect of KB-R, a specific NCX inhibitor for Ca(2+) in the influx mode. Alterations in extracellular [Na] in the saline did not affect intracellular Ca(2+) transport. Caffeine, responsible for inducing Ca release from sarcoplasmic reticulum in vertebrate muscle, increased intracellular Ca(2+) 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 Ca(2+) 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 Ca(2+) homeostasis through various Ca(2+) intracellular sequestration mechanisms and/or plasma membrane Ca(2+) influx and outflux that are highly regulatory. In summary, studies using whole gill cells are an interesting approach for working with real regulatory Ca(2+) mechanisms in intact cells under physiological Ca levels (mM range), compared to earlier work using isolated vesicles of various epithelial cells.