How does cAMP increase active Na absorption across alveolar epithelium?

AMILORIDE-SENSITIVE ACTIVE TRANSPORT of Na across the alveolar epithelium removes lung liquid in the neonate and protects against edema in the adult. Several lines of evidence have established that this process is stimulated by b-adrenergic agents via elevation of cAMP. First, when the lungs are filled with saline in vivo, then b-adrenergic agents or exogenous cAMP stimulates removal of this liquid (2, 3, 12). Furthermore, the increase in liquid clearance induced by terbutaline is inhibited by amiloride (3, 12). Second, in cultured cells, the amiloride-sensitive short-circuit current (Isc) is stimulated by terbutaline (4, 10). Finally, the terbutaline-induced increase in Isc is mirrored by an increase in net Na absorption as determined from the transepithelial fluxes of 22Na (7). Active Na absorption across the alveolar epithelium presumably occurs by the standard mechanism (8): diffusion of Na across the apical membrane down its electrochemical gradient via amiloride-sensitive channels followed by active extrusion across the basolateral membrane by Na-K-ATPase. The inward Na current across the apical membrane (INa) is given by the product of the membrane conductance to Na (GNa) and the driving force for net Na entry (Va 2 ENa), where Va is the apical membrane potential difference and ENa is the equilibrium potential for Na. In this issue of the American Journal of Physiology-Lung Cellular and Molecular Physiology, Lazrak et al. (9) argue that b-adrenergic agents stimulate Na absorption by an effect on GNa, whereas O’Grady et al. (11) believe that the stimulation is due to a change in Va brought about by an increase in Cl conductance. The overall Na conductance of the alveolar apical membrane (in mS/cm2) is given by GNa 5 g ·Po ·N, where g is the conductance of individual channels, Po is their average probability of being open, and N is their frequency per unit area of membrane. Patch-clamp studies on cultured type II cells (9) have shown that the predominant Na channel is amiloride sensitive and has a G of ,27 pS. Terbutaline approximately doubles the Po of this channel without affecting G (9). The percent increase in Po of this channel is approximately the same as the percent increase in active Na absorption measured as amiloride-sensitive Isc in Ussing chambers. However, these experiments were done on single isolated cells, not confluent cell sheets with tight junctions and a measurable transepithelial resistance. It is argued (9) that ‘‘ATII cells in primary culture orient themselves so that their basal membranes are attached to the substratum and the apical membranes are pointing upward.’’ But without tight junctions, one cannot be sure that the upper membrane has the same composition as a true apical membrane. In fact, it almost certainly doesn’t. Structural specializations on the apical membranes of confluent polarized cells (e.g., microvilli and glycocalyx) make them notoriously hard to patch clamp, which is why most studies are performed on single cells or small clumps of cells. Extrapolation of the results from single cells to the function of the intact epithelium should be done with caution; absence of tight junctions may alter the types of ion transport present. In airway epithelium, for instance, the Na-K-2Cl cotransporter of intact epithelium becomes a Na-Cl cotransporter on cell dispersion (5), and nonconfluent cells contain a form of Ca-activated Cl conductance absent from the apical membrane of intact epithelia (1). In contrast to a direct opening of Na channels, O’Grady et al. (11) suggest an indirect role for Cl in modulating active absorption of Na. Specifically, they propose that the apical membrane of alveolar type II cells has a cAMP-dependent Cl conductance. Also, the electrochemical gradient for Cl is inward across the apical membrane (i.e., ECl is more negative than Va). Therefore, the opening of Cl channels by cAMP results in hyperpolarization of the apical membrane and an increased driving force for entry of Na. This is plausible, but close inspection of the original article by Jiang et al. (6) shows that terbutaline did not in fact increase overall Isc nor are data presented that show that terbutaline increases amiloride-sensitive Isc. In fact, representative traces suggest that terbutaline did not increase active Na absorption (see Fig. 1 in Ref. 11). Nevertheless, others using slightly different alveolar type II cell cultures have reported increases of 40 (4) or 110% (10) in active Na absorption with terbutaline, and it would be worth using such cells to test the mechanism proposed by O’Grady et al. (11). The first step of O’Grady et al.’s hypothesis has been established: cultures of alveolar cells do indeed have an apical membrane Cl conductance. Thus Jiang et al. (6) permeated the basolateral membrane of confluent cell sheets with amphotericin and imposed a Cl gradient across the remaining apical membrane. The addition of terbutaline or cAMP to such preparations increased the Isc. The authors used the same permeabilized preparation to provide evidence against a cAMP-activated apical GNa. With a Na gradient imposed across the isolated Am. J. Physiol. Lung Cell. Mol. Physiol. 278: L231–L232, 2000.