Active chloride transport in the <i>in vitro</i> opercular skin of a teleost (<i>Fundulus heteroclitus</i>), a gill‐like epithelium rich in chloride cells

111

SUMMARY Na+/K+-ATPase,Na+/K+/2Cl- cotransporter (NKCC) and cystic fibrosis transmembrane conductance regulator (CFTR) are the three major transport proteins thought to be involved in chloride secretion in teleost fish. If this is the case, the levels of these transporters should be high in chloride cells of seawater-acclimated fish. We therefore examined the influence of salinity on immunolocalization of Na+/K+-ATPase, NKCC and CFTR in the gills of the Hawaiian goby (Stenogobius hawaiiensis). Fish were acclimated to freshwater and 20‰ and 30‰ seawater for 10 days. Na+/K+-ATPase and NKCC were localized specifically to chloride cells and stained throughout most of the cell except for the nucleus and the most apical region, indicating a basolateral/tubular distribution. All Na+/K+-ATPase-positive chloride cells were also positive for NKCC in all salinities. Salinity caused a slight increase in chloride cell number and size and a slight decrease in staining intensity for Na+/K+-ATPase and NKCC, but the basic pattern of localization was not altered. Gill Na+/K+-ATPase activity was also not affected by salinity. CFTR was localized to the apical surface of chloride cells, and only cells staining positive for Na+/K+-ATPase were CFTR-positive. CFTR-positive cells greatly increased in number (5-fold), area stained (53%) and intensity (29%)after seawater acclimation. In freshwater, CFTR immunoreactivity was light and occurred over a broad apical surface on chloride cells, whereas in seawater there was intense immunoreactivity around the apical pit (which was often punctate in appearance) and a light subapical staining. The results indicate that Na+/K+-ATPase, NKCC and CFTR are all present in chloride cells and support current models that all three are responsible for chloride secretion by chloride cells of teleost fish.

Section: Discussionsupporting

confidence: 59%

Influence of salinity on the localization of Na+/K+-ATPase,Na+/K+/2Cl- cotransporter (NKCC) and CFTR anion channel in chloride cells of the Hawaiian goby (Stenogobius hawaiiensis)

McCormick

Sundell

Björnsson

et al. 2003

111

“…For example, in F. heteroclitus, TEP was +18-23 mV, while the calculated E Na was +28 mV (Pic, 1978). It is well known that stress-generated catecholamines are potent inhibitors of active Cl − secretion in OE (Degnan et al, 1977;Foskett et al, 1982;Marshall and Bern, 1980) and this approach is a way of estimating the electrogenic component of the measured voltage, suggesting that 21 mV of the total TEP was electrogenic. Recently, confirming these estimates, TEP measured in SW for adult mummichogs was found to be +23 mV compared with the Nernst E Na of +29.1 mV (Wood and Grosell, 2008).…”

Section: Functional Interpretationmentioning

confidence: 92%

“…The in vitro mummichog OE bathed in symmetrical saline develops a V t of 15-30 mV, a transepithelial potential that is strongly inhibited by addition of the α-adrenergic agonist clonidine (Marshall et al, 1993) and by the loop diuretics furosemide and bumetanide (Degnan et al, 1977;Eriksson and Wistrand, 1986) and here V t might be taken as being mostly electrogenic, in that full inhibition in symmetrical saline brings the V t close to zero. In these conditions, Na + influx and efflux respond to voltage clamping (+ and −25 mV) and conform to the predicted flux ratio equation, all indicating that Na + moves through a single barrier to Na + diffusion (Degnan and Zadunaisky, 1980).…”

Section: Functional Interpretationmentioning

confidence: 99%

“…Transepithelial potential difference (V t ; mV) was measured continuously; pulses of current (ΔI=10 µA) were delivered 1.0 s once per minute, producing voltage transients (ΔV t ) and allowing calculation of OE resistance (R t =ΔV t /ΔI×0.125 Ω cm 2 ) and OE current (I sc =V t /R µA cm −2 ); output was recorded (iWorx ® data recorder) with LabScribe ® software. The I sc has been shown to be equivalent to the net tracer flux of Cl − across the OE or skin from seawater teleost fish mummichog (Degnan et al, 1977) and Gillichthys mirabilis skin (Marshall, 1981) when saline bathed both sides of the epithelium. To test asymmetrical conditions, isotonic saline on the mucosal side was replaced by full-strength SW and, to test hypersaline conditions, 2SW on the mucosal side.…”

Section: Electrophysiology Using Ussing Chambersmentioning

confidence: 99%

See 1 more Smart Citation

Paracellular pathway remodeling enhances sodium secretion by teleost fish in hypersaline environments

Cozzi

Robertson

Spieker

et al. 2015

In vertebrate salt-secreting epithelia, Na + moves passively down an electrochemical gradient via a paracellular pathway. We assessed how this pathway is modified to allow Na + secretion in hypersaline environments. Mummichogs (Fundulus heteroclitus) acclimated to hypersaline [2× seawater (2SW), 64‰] for 30 days developed invasive projections of accessory cells with an increased area of tight junctions, detected by punctate distribution of CFTR (cystic fibrosis transmembrane conductance regulator) immunofluorescence and transmission electron miscroscopy of the opercular epithelia, which form a gill-like tissue rich in ionocytes. Distribution of CFTR was not explained by membrane raft organization, because chlorpromazine (50 μmol l ) did not affect opercular epithelia electrophysiology. Isolated opercular epithelia bathed in SW on the mucosal side had a transepithelial potential (V t ) of +40.1±0.9 mV (N=24), sufficient for passive Na + secretion (Nernst equilibrium voltage≡E Na =+24.11 mV). Opercular epithelia from fish acclimated to 2SW and bathed in 2SW had higher V t of +45.1±1.2 mV (N=24), sufficient for passive Na + secretion (E Na =+40.74 mV), but with diminished net driving force. Bumetanide block of Cl − secretion reduced V t by 45% and 29% in SW and 2SW, respectively, a decrease in the driving force for Na + extrusion. Estimates of shunt conductance from epithelial conductance (G t ) versus short-circuit current (I sc ) plots (extrapolation to zero I sc ) suggested a reduction in total epithelial shunt conductance in 2SW-acclimated fish. In contrast, the morphological elaboration of tight junctions, leading to an increase in accessory-cell-ionocyte contact points, suggests an increase in local paracellular conductance, compensating for the diminished net driving force for Na + and allowing salt secretion, even in extreme salinities.

“…In the membrane chambers, the following epithelial electrophysiological variables were monitored: transepithelial potential V t (mV), transepithelial resistance R t (Ω·cm 2 ) and short-circuit current I sc ·µAcm -2 ). I sc is expressed as positive for secretion of anions and is equivalent to the net secretion of Cl - (Degnan et al, 1977). Epithelia were left at open circuit and were clamped to 0 mV for short periods to allow recording of I sc .…”

Section: Electrophysiologymentioning

confidence: 99%

Hypotonic shock mediation by p38 MAPK, JNK, PKC, FAK, OSR1 and SPAK in osmosensing chloride secreting cells of killifish opercular epithelium

Marshall

Ossum

Hoffmann

2005

110

SUMMARY Hypotonic shock rapidly inhibits Cl- secretion by chloride cells, an effect that is osmotic and not produced by NaCl-depleted isosmotic solutions, yet the mechanism for the inhibition and its recovery are not known. We exposed isolated opercular epithelia, mounted in Ussing chambers, to hypotonic shock in the presence of a variety of chemicals: a general protein kinase C (PKC) inhibitor chelerythrine, Gö6976 that selectively blocks PKCα and β subtypes, H-89 that blocks PKA, SB203580 that blocks p38 mitogen-activated protein kinase (MAPK), as well as serine/threonine protein phosphatase (PP1 and 2A) inhibitor okadaic acid, and finally tamoxifen, a blocker of volume-activated anion channels (VSOAC). Chelerythrine has no effect on hypotonic inhibition but blocked the recovery, indicating PKC involvement in stimulation. Gö6976 had little effect, suggesting that PKCα and PKCβ subtypes are not involved. H-89 did not block hypotonic inhibition but decreased the recovery, indicating PKA may be involved in the recovery and overshoot (after restoration of isotonic conditions). SB203580 significantly enhanced the decrease in current by hypotonic shock, suggesting an inhibitory role of p38 MAPK in the hypotonic inhibition. Okadaic acid increased the steady state current, slowed the hypotonic inhibition but made the decrease in current larger; also the recovery and overshoot were completely blocked. Hypotonic stress rapidly and transiently increased phosphorylated p38 MAPK (pp38) MAPK (measured by western analysis) by eightfold at 5 min, then more slowly again to sevenfold at 60 min. Hypertonic shock slowly increased p38 by sevenfold at 60 min. Phosphorylated JNK kinase was increased by 40-50% by both hypotonic and hypertonic shock and was still elevated at 30 min in hypertonic medium. By immunoblot analysis it was found that the stress protein kinase (SPAK) and oxidation stress response kinase 1 (OSR1) were present in salt and freshwater acclimated fish with higher expression in freshwater. By immunocytochemistry,SPAK, OSR1 and phosphorylated focal adhesion kinase (pFAK) were colocalized with NKCC at the basolateral membrane. The protein tyrosine kinase inhibitor genistein (100 μmol l-1) inhibited Cl- secretion that was high, increased Cl- secretion that was low and reduced immunocytochemical staining for phosphorylated FAK. We present a model for rapid control of CFTR and NKCC in chloride cells that includes: (1) activation of NKCC and CFTR via cAMP/PKA, (2) activation of NKCC by PKC, myosin light chain kinase (MLCK), p38, OSR1 and SPAK, (3) deactivation of NKCC by hypotonic cell swelling, Ca2+ and an as yet unidentified protein phosphatase and (4) involvement of protein tyrosine kinase (PTK) acting on FAK to set levels of NKCC activity.