A novel role for cyclic nucleotide-gated cation channels in lung liquid homeostasis in sheep

Abstract

1. Sheep lungs were artificially perfused in situ with warmed whole oxygenated sheep blood. The airspaces of the lungs were filled with liquid containing an impermeant tracer, to allow measurement of the rate of net transepithelial liquid movement under various conditions. 2. Dichlorobenzamil (1.5 x 10-5 M), a blocker of cyclic nucleotide-gated cation channels, inhibited the resting absorption of lung liquid in sheep aged 6 months (n = 5) (from -36.47 +/- 4.62 to -4.36 +/- 5.27 ml h-1, means +/- s.e.m.; P < 0.005, paired t test). Amiloride (10-4 M), a blocker of epithelial sodium channels, had no additive effect to that of dichlorobenzamil. 3. In the lungs of sheep aged 6 months (n = 4), amiloride (10-4 M) partially inhibited the resting absorption of liquid (from -35.21 +/- 8.57 to -11.05 +/- 4.91 ml h-1; P < 0.05, one-tailed paired t test), and dichlorobenzamil (1.5 x 10-5 M) exerted an additive effect to that of amiloride resulting in secretion at +6.29 +/- 3.05 ml h-1 (P < 0. 01, paired t test). 4. In the lungs of sheep aged 6 weeks (n = 3), amiloride (10-4 M) also inhibited the resting absorption of liquid (from -26.36 +/- 14.05 to -5.17 +/- 8.27 ml h-1; P < 0.05, one-tailed paired t test); however, dichlorobenzamil (1.5 x 10-5 M) did not exert an additive effect to that of amiloride. 5. In the lungs of sheep aged 6 months (n = 4), amiloride (10-4 M) partially inhibited the resting absorption of liquid (from -35.70 +/- 8.58 to -6.79 +/- 4.28 ml h-1; P < 0.05, paired t test), and pimozide (1.5 x 10-4 M), another blocker of cyclic nucleotide-gated cation channels, also exerted an additive effect to that of amiloride, resulting in secretion of lung liquid at +15.36 +/- 9.14 ml h-1 (P < 0.05, paired t test). 6. We conclude that cyclic nucleotide-gated cation channels mediate a component of lung liquid absorption in sheep aged 6 months (but not in sheep aged 6 weeks), and that a mechanism for lung liquid secretion (present in fetuses) is retained at 6 months of age.

Figure 1. Example experiments performed on the artificially perfused in situ lungs of sheep aged 6 months (A, B and D) or 6 weeks (C)

J_v (in ml h⁻¹) is given above the regression line for each intervention period. Unpaired t tests were used to compare regression lines. A, dichlorobenzamil was administered at two doses in order to produce a dose-response curve (data not shown): 1.5 × 10⁻⁶ M dichlorobenzamil (first arrow) caused a significant reduction in J_v relative to the control period (* P < 0.01); 1.5 × 10⁻⁵ M dichlorobenzamil (second arrow) had no significant effect relative to the rate of absorption in the presence of 1.5 × 10⁻⁶ M dichlorobenzamil. Amiloride at 10⁻⁴ M had no significant effect relative to absorption in the presence of 1.5 × 10⁻⁵ M dichlorobenzamil or 1.5 × 10⁻⁶ M dichlorobenzamil. B, amiloride (10⁻⁴ M) caused a significant reduction in J_v relative to the control period (* P < 0.001). Dichlorobenzamil (1.5 × 10⁻⁵ M) had an additive effect relative to that of amiloride (** P < 0.05). C, amiloride (10⁻⁴ M) caused a significant reduction in the control rate of absorption (* P < 0.001). The rate of absorption in the presence of dichlorobenzamil (1.5 × 10⁻⁵ M) was not significantly different to that in the presence of amiloride (P > 0.1). D, amiloride (10⁻⁴ M) caused a significant reduction in J_v relative to the control period (* P < 0.05). Pimozide (1.5 × 10⁻⁴ M) had an additive effect relative to that of amiloride (** P < 0.05).