Control of Cl-transport in the operculum epithelium of Fundulus heteroclitus : long- and short-term salinity adaptation

AbstractThe eurohaline fish, Fundulus heteroclitus, adapts rapidly to enhanced salinity by increasing the ion secretion by gill chloride cells. An increase of ∼70 mOsm in plasma osmolarity was previously found during the transition. To mimic this in vitro, isolated opercular epithelia of seawater-adapted Fundulus mounted in a modified Ussing chamber were exposed to an increase in NaCl and/or osmolarity on the basolateral side, which immediately increased ISC. Various Cl− channel blockers as well as the K+ channel blocker Ba2+ added to the basolateral side all inhibited the steady-state as well as the hypertonic stimulation of ISC. The ∃-agonist isoproterenol stimulates ISC in standard Ringer solutions. In contrast, when cell volume was kept at the larger value by simultaneous addition of water, the stimulation with isoproterenol was abolished, suggesting that the key process for activation of the Na+, K+, 2Cl− cotransporter is cell shrinkage. The protein kinase C (PKC) inhibitor chelerythrine and the myosin light chain kinase (MLCK) inhibitor ML-7 had strong inhibitory effects on the mannitol activation of ISC, thus both MLCK and PKC are involved. The two specific protein kinase A (PKA) inhibitors H-89 and KT 5720 had no effect after mannitol addition whereas isoproterenol stimulation was completely blocked by H-89. This indicates that PKA is involved in the activation of the apical Cl− channel via c-AMP whereas the shrinkage activation of the Na+, K+, 2Cl− cotransporter is independent of PKA activation. The steady-state Cl− secretion was stimulated by an inhibitor of serine/threonine phosphatases of the PP-1 and PP-2A type and inhibited by a PKC inhibitor but not by a PKA inhibitor. Thus, it seems to be determined by continuous phosphorylation and dephosphorylation involving PKC but not PKA. The steady-state Cl− secretion and the maximal obtainable Cl− secretion were measured in freshwater-adapted fish and in fish retransferred to saltwater. No ISC could be measured in freshwater-adapted fish or in the fish within the first 18 h after transfer to saltwater. As evidenced from Western blot analysis using antiserine-antibodies, a heavily serine phosphorylated protein of about 190 kDa was consistently observed in the saltwater-acclimated fish, but was only weakly present in freshwater-acclimated fish. This observation indicates that acclimatization to saltwater stimulates the expression of this 190-kDa protein and/or a serine/threonine kinase, which subsequently phosphorylates the protein

Osmoregulation in the mudskipper, Boleophthalmus boddaerti I. Responses of branchial cation activated and anion stimulated adenosine triphosphatases to changes in salinity

10.1007/BF01987612Fish Physiology and Biochemistry9163-68FPBI

The Na+-K+-2Cl- cotransporter and the osmotic stress response in a model salt transport epithelium.

Epithelia are physiologically exposed to osmotic stress resulting in alteration of cell volume in several aspects of their functioning; therefore, the activation of ‘emergency’ systems of rapid cell volume regulation is fundamental in their physiology. In this review, the physiological response to osmotic stress, particularly hypertonic stress, was described in a salt-transporting epithelium, the intestine of the euryhaline teleost European eel. This epithelium is physiologically exposed to changes in extracellular osmolarity and represents a good physiological model for functional studies on cellular volume regulation, permitting the study of volume regulated ion transport mechanisms in a native tissue. An absorptive form of the cotransporter, homologue of the renal NKCC2, localized on the apical membrane, was found in the intestine of the euryhaline teleost European eel. This cotransporter accounts for the luminal uptake of Cl); it operates in series with a basolateral Cl) conductance and presumably a basolateral electroneutral KCl cotransport and in parallel with a luminal K+ conductance. The ion transport model described for eel intestine, based on the operation of an absorptive luminal Na+–K+– 2Cl), is basically the same as the model that has been proposed for the thick ascending limb (cTAL) of the mammalian renal cortex. This paper focuses on the role of Na+–K+–2Cl) cotransport in the responses to hypertonic stress in the eel intestine and the role of cytoskeleton (either actin-based or tubulin based) is discussed