CA2+ TRANSPORT BY OPERCULAR EPITHELIUM OF THE FRESH-WATER-ADAPTED EURYHALINE TELEOST, FUNDULUS-HETEROCLITUS

We examined transepithelial transport of Ca2+ across the isolated opercular epithelium of the euryhaline killifish adapted to fresh water. The opercular epithelium, mounted in vitro with saline on the serosal side and fresh water (0.1 mmol . l(-1) Ca2+) bathing the mucosal side, actively transported Ca2+ in the uptake direction; net flux averaged 20-30 nmol . cm(-2). h(-1). The rate of Ca2+ uptake varied linearly with the density of mitochondria-rich cells in the preparations. Ca2+ uptake was saturable, apparent K-1/2 of 0.348 mmol . l(-1), indicative of a multistep transcellular pathway. Ca2+ uptake was inhibited partially by apically added 0.1 mmol . l(-1) La3+ and 1.0 mmol . l(-1) Mg2+. Addition of dibutyryl-cyclic adenosine monophosphate (0.5 mmol . l(-1)) + 0.1 mmol . l(-1) 3-isobutyl-1-methylxanthine inhibited Ca2+ uptake by 54%, but epinephrine, clonidine and isoproterenol were without effect. Agents that increase intracellular Ca2+, thapsigargin (1.0 mu mol . l(-1), serosal side), ionomycin (1.0 mu mol . l(-1), serosal side) and the calmodulin blocker trifluoperazine (50 mu mol . l(-1), mucosal side) all partially inhibited Ca2+ uptake. In contrast, apically added ionomycin increased mucosal to serosal unidirectional Ca2+ flux, indicating Ca2+ entry across the apical membrane is rate limiting in the transport. Verapamil (10-100 mu mol . l(-1), mucosal side), a Ca2+ channel blocker, had no effect. Results are consistent with a model of Ca2+ uptake by mitochondria rich cells that involves passive Ca2+ entry across the apical membrane via verapamil-insensitive Ca2+ channels, intracellular complexing of Ca2+ by calmodulin and basolateral exit via an active transport process. Increases in intracellular Ca2+ invoke a downregulation of transcellular Ca2+ transport, implicating Ca2+ as a homeostatic mediator of its own transport.