Using pH- and voltage-sensitive microelectrodes, as well as the two-electrode voltage-clamp and macropatch techniques, we compared the functional properties of the three NBCe1 variants (NBCe1-A, -B, and -C) with different amino and/or carboxy termini expressed in Xenopus laevis oocytes. Oocytes expressing rat brain NBCe1-B and exposed to a CO2/HCO3 − solution displayed all the hallmarks of an electrogenic Na+/HCO3 − cotransporter: (a) a DIDS-sensitive pHi recovery following the initial CO2-induced acidification, (b) an instantaneous hyperpolarization, and (c) an instantaneous Na+-dependent outward current under voltage-clamp conditions (−60 mV). All three variants had similar external HCO3 − dependencies (apparent KM of 4–6 mM) and external Na+ dependencies (apparent KM of 21–36 mM), as well as similar voltage dependencies. However, voltage-clamped oocytes (−60 mV) expressing NBCe1-A exhibited peak HCO3 −-stimulated NBC currents that were 4.3-fold larger than the currents seen in oocytes expressing the most dissimilar C variant. Larger NBCe1-A currents were also observed in current–voltage relationships. Plasma membrane expression levels as assessed by single oocyte chemiluminescence with hemagglutinin-tagged NBCs were similar for the three variants. In whole-cell experiments (Vm = −60 mV), removing the unique amino terminus of NBCe1-A reduced the mean HCO3 −-induced NBC current 55%, whereas removing the different amino terminus of NBCe1-C increased the mean NBC current 2.7-fold. A similar pattern was observed in macropatch experiments. Thus, the unique amino terminus of NBCe1-A stimulates transporter activity, whereas the different amino terminus of the B and C variants inhibits activity. One or more cytosolic factors may also contribute to NBCe1 activity based on discrepancies between macropatch and whole-cell currents. While the amino termini influence transporter function, the carboxy termini influence plasma membrane expression. Removing the entire cytosolic carboxy terminus of NBCe1-C, or the different carboxy terminus of the A/B variants, causes a loss of NBC activity due to low expression at the plasma membrane.

In the United States, mycoplasmas account for up to 20 to 30% of the 4 million cases of pneumonia reported annually and cause significant morbidity and mortality. Murine respiratory infection with Mycoplasma pulmonis reproduces the essential features of human respiratory mycoplasmosis. This model has revealed that reactive oxygen and nitrogen species (RONS) produced by the alveolar macrophage (AM) and surfactant protein A, secreted by the alveolar type II (ATII) cells, are essential for early clearance of respiratory mycoplasmas in vivo and in vitro (1, 2). However, even with significant production of RONS in response to mycoplasma infection, susceptible individuals may develop serious illness. In this context, adherence of mycoplasma species to the respiratory epithelium is necessary for effective coloniza-

We investigated the mechanisms of endogenous nitric oxide (NO) modulation of lung sodium (Na(+)) transport. C57BL/6 mice injected intraperitoneally with the specific inducible NO synthase (iNOS) inhibitor 1400W (10 mg/kg every 8 h for 72 h) exhibited decreased alveolar nitrite levels and Na(+)-dependent amiloride-sensitive alveolar fluid clearance as compared with mice injected with vehicle. Similarly, pretreatment of mouse tracheal epithelial cells with 1400W abolished the inhibitory effects of amiloride on their Na(+) short circuit currents. On the other hand, mouse tracheal epithelial cells pretreated with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a specific inhibitor of guanylate cyclase, had lower levels of cGMP, but normal values of amiloride-sensitive Na(+) currents. Amiloride also inhibited whole-cell Na(+) currents across A549 cells treated with vehicle (K(i) = 249 nM), but had no effect in A549 cells treated with 1400W. Western blotting studies showed significantly lower levels of alpha and gammaENaC in lung tissues and alveolar type II (ATII) cells from iNOS(-/-) as well as iNOS(+/+) mice treated with 1400W, as compared with the corresponding values from vehicle-treated iNOS(+/+) mice. Similar values for ratios of alpha, beta, and gammaenac to gapdh were obtained by real-time polymerase chain reaction for iNOS(+/+) mice and iNOS(-/-) mice. We concluded that NO derived from iNOS under basal conditions is necessary for amiloride-sensitive Na(+) transport across lung epithelial cells and modulates the amount of alpha and gammaENaC via post-transcriptional, cGMP-independent mechanisms.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that undergoes endocytosis through clathrin-coated pits. Previously, we demonstrated that Y1424A is important for CFTR endocytosis (Prince, L. S., Peter, K., Hatton, S. R., Zaliauskiene, L., Cotlin, L.

A basally-active, Gd(3+)- and GsMTx-4-sensitive SAC current that is non-selective for Na(+) and K(+), but impermeable to Ca(2+) under resting conditions is transiently elevated in LV myocytes from rats in early stages of volume overload-induced HF.