Re-evaluating the Na+ conductance of adult rat alveolar type II pneumocytes:: evidence for the involvement of cGMP-activated cation channels

1. Alveolar epithelial type II pneumocytes were isolated and purified from adult rat lung by elastase digestion and differential adhesion, and cultured in serum-free medium for similar to2 days on glass coverslips for subsequent patch-clamp studies employing symmetric also diumisethionate solutions. 2.Whole-cell Na(+)currents exhibited essentially linear current-voltage relationships which were mildly inhibited (by similar to 25 %) by 10 muM amiloride. In contrast, 1 mM Zn2+ inhibited the currents by similar to 55 % with an IC50 of similar to 134 muM and maximal blockade achieved between 5 and 10 mM. The effects of Zn2+ and amiloride were additive, and independent of the order of blocker addition. 3. Gd2+, Zn2+ and La3+ at 10 mM were all effective at rapidly, reversibly and significantly blocking the amiloride-insensitive currents by similar to 60%. In contrast, Ni2+ was a very weak inhibitor (30% inhibition at 10 rn Ni). 4. Pimozide (10 muM) caused inhibition of whole-cell cation conductance by similar to 55 %. The inhibitory effect of pimozide was concentration dependent with an IC50 of -1 mum and was maximally effective between 10 and 30 muM. Sequential addition of Zn2+ and pimozide, in either order, revealed no overlapping inhibitory effect on the amiloride-insensitive conductance, and supported the notion that the Zn--(2+) and pimozide-sensitive currents are identical. 5. The amiloride-insensitive, Zn(-)(2+)blockable conductance was characterised by a Na+/K+ permeability ratio (P-Na/P-K) of 0.73 +/- 0.02. 6. 8Br-cGMP (100 muM), a membrane-permeable analogue of cGMP, evoked a robust activation of whole-cell cation conductance to 220 % of control. This activation was apparent in either the absence or the presence of 10 muM, amiloride, but was completely abolished in the presence of Zn2+. 7. These data support the in vivo and in situ observations of a substantial amiloride-resistant Na+ conductance, demonstrate directly that cyclic nucleotide-gated non-selective cation channels are functionally expressed in alveolar epithelial type II cells, and suggest that these channels may contribute to the fluid-reabsorptive driving force in adult lung.