Focal adhesion kinase and osmotic responses in ionocytes of Fundulus heteroclitus, a euryhaline teleost fish.

Cystic Fibrosis Transmembrane conductance Regulator (CFTR) anion channels are the regulated exit pathway in Cl- secretion by teleost salt secreting ionocytes of the gill and opercular epithelia of euryhaline teleosts. By confocal light immunocytochemistry using regular and phospho-antibodies directed against conserved sites, we found that killifish CFTR (kfCFTR) and the tyrosine kinase Focal Adhesion Kinase (FAK) phosphorylated at Y407 (FAKpY407) and FAKpY397 are colocalized at the apical membrane and in subjacent membrane vesicles of ionocytes. Hypotonic shock and the α-2 adrenergic agonist clonidine rapidly and reversibly inhibit Cl- secretion by isolated opercular epithelia, simultaneous with dephosphorylation of FAKpY407 and increased FAKpY397, located in the apical membrane of ionocytes in the opercular epithelium. FAKpY407 is re-phosphorylated at the apical membrane of ionocytes and Cl- secretion rapidly restored by hypertonic shock, detectable at 2 min., maximum at 5 min and still elevated at 30 min. In isolated opercular epithelia, the FAK phosphorylation inhibitor Y15 and p38MAP kinase inhibitor SB203580 significantly blunted the recovery of short-circuit current (Isc, equal to Cl- secretion rate) after hypertonic shock. The cSRC inhibitor saracatinib dephosphorylated FAKpY861 seen near tight junctions of pavement cells, and reduced the increase in epithelial resistance normally seen with clonidine inhibition of ion transport, while FAKpY397 was unaffected. The results show rapid osmosensitive responses in teleost fish ionocytes involve phosphorylation of CFTR by FAKpY407, an opposing role for FAKpY397 and a possible role for FAKpY861 in tight junction dynamics.

Cold acclimation of NaCl secretion in a eurythermic teleost: mitochondrial function and gill remodeling.

Active chloride secretion, measured as short-circuit current (Isc) in ionocytes of opercular epithelia (OE) in the eurythermic, euryoxic, and euryhaline killifish or mummichog (Fundulus heteroclitus) was studied in cold (5°C) and warm (20°C) acclimated fish to determine if homeoviscous adaptation aided chloride secretion in the cold. Isolated opercular epithelia were cooled from 30°C to 0.2°C for warm and cold acclimated fish; from 30 to 8°C, Isc decreased with Q10=1.68 for warm and Q10=1.56 for cold acclimated tissues. By Arrhenius plots, there is a critical temperature, 8°C, below which aerobic Isc decreased sharply (Q10=6.90 for warm and 4.23 for cold acclimated tissues), suggesting a shift in mitochondrial efficiency of oxidative phosphorylation. In anaerobic conditions (0.5mM NaCN; N2 saturation), chloride transport continued at a lower rate, and Isc decrease with cooling below 8°C was less pronounced (Q10=2.95 for warm and 3.08 for cold), suggesting a shift in transporter function in plasma membrane. Under anaerobic conditions, NaCl secretion at 20°C was reversibly inhibited by hypotonic shock, indicating normal regulation of transport. Chloride secretion in warm-acclimated fish was supported mostly (75% at 20°C) by aerobic metabolism, whereas that for cold-acclimated fish was lower (55% at 20°C), suggesting a greater reliance on anaerobic metabolism in the cold. Once acclimated to cold, ionocytes may be temporarily incapable of increasing their aerobic ATP supply, even when warmed to 30°C. In cold acclimated fish there was increased polyunsaturated fatty acid composition of gill epithelium (consistent with homeoviscous adaptation) and gill remodeling, wherein epithelial cells filled the interlamellar space (interlamellar cell mass, ILCM) by as much as 70%, thus increasing diffusion distance against passive ion gain. Most ionocytes in these thickened epithelial masses became taller, still connecting basal lamina with the environment, consistent with the continuing transport rates at low temperatures. Whereas the low aerobic scope of cold-acclimated fish and thickened gill epithelium is appropriate to winter inactivity, metabolic depression and anaerobiosis, the large aerobic scope of warm-acclimated fish favors active foraging at high temperatures.