Evidence for Na-K-Cl cotransport in alveolar epithelial cells: Effect of phorbol ester and osmotic stress

Abstract

We have investigated the presence of Na-K-Cl cotransport in alveolar type II cells using uptake of 86Rb. Several data support the presence of a Na-K-Cl cotransport in these cells. First, a large fraction of ouabain-resistant 86Rb uptake was inhibited by bumetanide and furosemide. Second, bumetanide-sensitive 86Rb uptake required the presence of Na+ and Cl- in the incubation medium; dependency on extracellular Na+ and K+ was hyperbolic, with a Km of 14.6 mM and 8.3 mM, respectively, while dependency on extracellular Cl- was sigmoidal, which suggests a 1:1:2 stoichiometry. Third, a fraction of amiloride-insensitive 22Na influx was deeply inhibited by bumetanide. 22Na influx was dependent on the presence of extracellular K+ and Cl-. Since Na-K-Cl activity dramatically decreased with time in culture, further characterization of the cotransport on polarized cells could not be performed. The phorbol ester PMA inhibited Na-K-Cl cotransport in a time- and concentration-dependent manner. This inhibition was mimicked by oleoylacetylglycerol, dioctanoylglycerol, and the diacylglycerol kinase inhibitor R59022, and was reversed by an antagonist of PKC, staurosporine. Since the Na-K-Cl cotransport has been reported to be involved in cell volume regulation, we investigated its modulation by changes in extracellular osmolarity. Na-K-Cl activity was increased after a two-step procedure: swelling in hypotonic medium followed by shrinking in hypertonic medium. Under these conditions, cotransport activity increased whenever PKC activity was up- or downregulated, which suggests that the cell volume-induced modulation of the cotransport is independent from the PKC activity. Though we were not able to determine the polarity of the cotransport, it may also be involved in the absorptive function of alveolar type II cells, and would provide an alternate pathway for sodium entry.

Modulation of Chloride Secretion Across the Pigmented Rabbit Conjunctiva

“…Recent studies using T84 cells suggested -myoinositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P % ) as a negative messenger in blocking Ca# + -regulated Cl − channels following the rise in intracellular Ca# + level (Ismailov et al, 1996 ;Vajanaphanich et al, 1994). Furthermore, PMA has also been shown to down regulate the basolateral Na + -K + -ATPase (Beron et al, 1997) and Na + -(K + )-2Cl − cotransporter (Clerici et al, 1995) in A6 and alveolar epithelia, respectively. These factors may also contribute to the down regulation of Cl − secretion after maximal stimulation induced by PMA.…”

Rat lung alveolar type II cell line maintains sodium transport characteristics of primary culture

“…acids (Clerici et al, 1990) and Na+-K'-2Cl- (Clerici et al, 1995), and is extruded across the basolateral side by a Na+, K'-ATPase. However, the use of primary cultures for AT11 studies is restricted by: (1) early dedifferentiation: within a few days, surfactant secretion decreases, cell surface binding characteristics change and Na' transport drops (Yue et al, 1993); and (2) heterogeneity of the material obtained through enzymatic digestion: AT11 are contaminated by macrophages and fibroblasts, which may interfere with measurement of transport.…”

Hypotonic Shock Modulates Na+ Current via a Cl- and Ca2+/Calmodulin Dependent Mechanism in Alveolar Epithelial Cells

“…Clearly, the response of alveolar epithelial cells to hypotonic shock is different from that of kidney cells which have to adapt their ionic transport to the changing tonicity of the tubular environment. Some studies have reported changes in Rb + uptake in response to different tonicity challenges in alveolar epithelial cells [44]. To the best of our knowledge, the present investigation is the first to describe the impact of hypotonicity on transepithelial current generated in rat alveolar epithelial cells.…”