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 μM (Ca
2+ change × initial intracellular Ca
−1 × 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.
相似文献