Effect of apical hyperosmotic sodium challenge and amiloride on sodium transport in human bronchial epithelial cells from cystic fibrosis donors.

Hypertonic saline (HS) inhalation therapy benefits cystic fibrosis (CF) patients [Donaldson SH, Bennet WD, Zeman KL, Knowles MR, Tarran R, Boucher RC. N Engl J Med 354: 241-250, 2006; Elkins MR, Robinson M, Rose BR, Harbour C, Moriarty CP, Marks GB, Belousova EG, Xuan W, Bye PT; the National Hypertonic Saline in Cystic Fibrosis (NHSCF) Study Group. N Engl J Med 354: 229-240, 2006]. Surprisingly, these benefits are long-lasting and are diminished by the epithelial Na(+) channel blocker amiloride (Donaldson SH, Bennet WD, Zeman KL, Knowles MR, Tarran R, Boucher RC. N Engl J Med 354: 241-250, 2006). Our aim was to explain these effects. Human bronchial epithelial (hBE) cells from CF lungs were grown in inserts and were used in three experimental approaches: 1) Ussing chambers to measure amiloride-sensitive short-circuit currents (INa); 2) continuous perfusion Ussing chambers; and 3) near "thin-film" conditions in which the airway surface of the inserts was exposed to a small volume (30 µl) of isosmotic or HS solution as the inserts were kept in their incubation tray and were subsequently used to measure INa under isosmotic conditions (near thin-film experiments; Tarran R, Boucher RC. Methods Mol Med 70: 479-492, 2002). HS solutions (660 mosmol/kgH2O) were prepared by adding additional NaCl to the isosmotic buffer. The transepithelial short-circuit current (ISC), conductance (GT), and capacitance (CT) were measured by transepithelial impedance analysis (Danahay H, Atherton HC, Jackson AD, Kreindler JL, Poll CT, Bridges RJ. Am J Physiol Lung Cell Mol Physiol 290: L558-L569, 2006; Singh AK, Singh S, Devor DC, Frizzell RA, van Driessche W, Bridges RJ. Methods Mol Med 70: 129-142, 2002). Exposure to apical HS inhibited INa, GT, and CT. The INa inhibition required 60 min of reexposure to the isosmotic solution to recover 75%. The time of exposure to HS required to inhibit INa was <2.5 min. Under near thin-film conditions, apical exposure to HS inhibited INa, but as osmotically driven water moved to the apical surface, the aqueous apical volume increased, leading to an amiloride-insensitive decrease in its osmolality and to recovery of INa that lagged behind the osmotic recovery. Amiloride significantly accelerated the recovery of INa following exposure to HS. Our conclusions are that exposure to HS inhibits hBE INa and that amiloride diminishes this effect.

Na(+)-dependent transport of taurine is found only on the abluminal membrane of the blood-brain barrier.

Luminal and abluminal plasma membranes were isolated from bovine brain microvessels and used to identify and characterize Na(+)-dependent and facilitative taurine transport. The calculated transmembrane potential was -59 mV at time 0; external Na(+) (or choline under putative zero-trans conditions) was 126 mM (T=25 °C). The apparent affinity constants of the taurine transporters were determined over a range of taurine concentrations from 0.24 µM to 11.4 µM. Abluminal membranes had both Na(+)-dependent taurine transport as well as facilitative transport while luminal membranes only had facilitative transport. The apparent K(m) for facilitative and Na(+)-dependent taurine transport were 0.06±0.02 µM and 0.7±0.1 µM, respectively. The Na(+)-dependent transport of taurine was voltage dependent over the range of voltages studied (-25 to -101 mV). The transport was over 5 times greater at -101 mV compared to when V(m) was -25 mV. The sensitivity to external osmolality of Na(+)-dependent transport was studied over a range of osmolalities (229 to 398 mOsm/kg H(2)O) using mannitol as the osmotic agent to adjust the osmolality. For these experiments the concentration of Na(+) was maintained constant at 50mM, and the calculated transmembrane potential was -59 mV. The Na(+)-dependent transport system was sensitive to osmolality with the greatest rate observed at 229 mOsm/kg H(2)O.

Effect of extracellular glucose and K+ on intracellular osmolytes and volume in a human kidney cell line.

The goal of this study was to assess the effect of extracellular glucose and K+ ((K)o) on the intracellular osmolyte content and cell volume maintenance and regulation in a human embryonic kidney cell line (tsA201a). Cell volume maintenance was studied by isotonic (313 +/- 5 mOsm) replacement of culture media by a glucose-free Ringer solution containing (in mM) 0, 3, 6, or 10 K+. Cell volume regulation was studied by exposing cells to hypotonic (250 +/- 5 mOsm) glucose-free Ringer solution containing the various (K)o. The results showed that: 1) intracellular osomlyte content (i.e. Na+, Cl-, Urea and free amino acids (FAA)) and cell volume increased when culture media was replaced with isotonic Ringer at all (K)o; 2) osmolyte content decreased with continuous exposure to isotonic Ringer at all (K)o but cell volume changes depended on (K)o. Volume recovery occurred at 6 and 10 mM K+; 3) exposure to hypotonic Ringer induced swelling at all (K)o followed by a reduction in measured intracellular osmolytes. Regulatory volume decrease occurred in 6 or 10 mM K+ but swelling continued in 0 or 3 mM K+; and 4) addition of ouabain produced swelling without recovery under iso- and hypotonic conditions. These results indicate that the removal of extracellular glucose produced a transient inhibition of the Na+/K+ ATPase resulting in a transient increase in the intracellular content of Urea, FAA and cell volume and (K)o regulated an as yet unidentified intracellular osmolyte.

Ca2+ /H+ exchange via the plasma membrane Ca2+ ATPase in skeletal muscle.

The aims of this work were to determine: 1) whether Ca2+ exit via the plasmalemmal Ca2+ ATPase (PMCA) is coupled to H+ entry via a Ca2+/H+ exchange; 2) whether operation of PMCA has an absolute requirement on external H+ (Ho); and 3) the stoichiometry and voltage-dependence of the Ca2+/H+ exchange. Barnacle muscle cells were used because of the ease with which they can be internally-perfused (e.g., with 45Ca), voltage-clamped and impaled with a pH electrode. Thus, the simultaneous measurement of plasmalemmal Ca2+ and H+ fluxes can be measured. The effects of Ho, intracellular ATP, PMCA blockers, and membrane potential (VM) were studied on PMCA-mediated Ca2+/H+ exchange. The results indicate that: i) Ca2+ efflux is promoted by external acidification, is accompanied by a membrane depolarization, and by an intracellular acidification greater than the one resulting from Ho "leak" and PMCA-mediated ATP hydrolysis; ii) Ho-dependent Ca2+ efflux is inhibited by PMCA blockers and by ATP depletion and is accelerated by membrane depolarization (~3 fold by 20 mV depolarization); iii) the coupling ratio of the Ca2+/H+ exchange depends on Ho: at an extracellular pH (pHo)=6.5, the ratio is 1Ca2+:~3H+; at pHo=8.2, Ca2+ efflux rate is 3 times slower and the ratio is 1Ca2+: <1H+.