8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate-Na stimulates human alveolar fluid clearance by releasing external Na+ self-inhibition of epithelial Na+ channels

Abstract

Salt absorption via alveolar epithelial Na(+) channels (ENaC) is a critical step for maintaining an airspace free of flooding. Previously, we found that 8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate-Na (CPT-cGMP) activated native and heterologous ENaC. To investigate the potential pharmacological relevance, we applied this compound intratracheally to human lungs and found that ex vivo alveolar fluid clearance was increased significantly. Furthermore, this compound eliminated self-inhibition in human lung H441 cells and in oocytes expressing human αβγ but not δβγ channels. To further elucidate this novel mechanism, we constructed mutants abolishing (β(ΔV348) and γ(H233R)) or augmenting (α(Y458A) and γ(M432G)) self-inhibition. The mutants eliminating self-inhibition lost their responses to CPT-cGMP, whereas those enhancing self-inhibition facilitated the stimulatory effects of this compound. CPT-cGMP was unable to activate a high P(o) mutant (β(S520C)) and plasmin proteolytically cleaved channels. Our data suggest that elimination of self-inhibition of αβγ ENaC may be a novel mechanism for CPT-cGMP to stimulate salt reabsorption in human lungs.

Figure 1.

Stimulation of human alveolar fluid clearance (AFC) by 8-(4-chlorophenylthio)-guanosine-3′,5′-cyclic monophosphate-Na (CPT-cGMP). AFC was measured by delivering 0.9% NaCl with 5% BSA into human lung lobes ex vivo. The instillate was collected after 60 minutes, and AFC was measured as described in Materials and Methods. (A) AFC in the presence and absence of amiloride (Amil) and CPT-cGMP. n = 15 for controls; n = 5 for other groups. **P < 0.01; ***P < 0.001. (B) Amiloride-inhibitable AFC fraction associated with ENaC. *P < 0.05.

Figure 2.

CPT-cGMP alters self-inhibition in human lung epithelial cells. (A) Self-inhibition traces recorded at −60 mV in H441 cells in the absence (Basal) and presence of CPT-cGMP and amiloride (right). (B) Sustained (I_sus)/maximal (I⁰_max) current ratio. *P < 0.05; n = 5.

Figure 3.

Activation of whole-cell and single-channel epithelial Na⁺ channel (ENaC) activities by CPT-cGMP. (A and B) Current traces of αβγ (A) and δβγ (B) ENaC channels digitized at the holding potentials of −100 mV and +80 mV. (C) Activation by CPT-cGMP as revealed by increased fold in current levels at −100 mV. ***P < 0.001. (D) Dose–response curves of αβγ ENaC. The median effective concentration values at −100 mV and +80 mV are shown. (E) Single-channel tracings in an outside-out patch. Membrane potential, −60 mV. (F) Single-channel activity before (Basal) and after perfusion of CPT-cGMP and amiloride (Amil). n = 6. **P < 0.01. (G) Single-channel tracings in a cell-attached patch.

Figure 4.

CPT-cGMP modifies self-inhibition of αβγ ENaC in oocytes. Self-inhibition was measured by fast switching the low Na⁺ bath solution (1 mM) to regular ND96 medium (96 mM). (A) Representative whole-cell current trace digitized at −60 mV. CPT-cGMP (0.2 mM) was added after the first three sweeps and washed out for the last three sweeps. (B) Current ratio. n = 9. *P < 0.05; **P < 0.01 versus the first sweep. (C) Comparison of CPT-cGMP on self-inhibition of αβγ and δβγ ENaC channels. Paired traces were recorded before and after application of CPT-cGMP.

Figure 5.

Responses of self-inhibition mutant to CPT-cGMP. (A) Representative current traces of mutants eliminating self-inhibition. Application of CPT-cGMP is indicated by horizontal lines. (B) Current tracings of mutants boosting self-inhibition. (C and D) Fold increased by CPT-cGMP for mutants abolishing (C) and facilitating self-inhibition (D). **P < 0.01 and ***P < 0.001 versus αβγ ENaC. n = 10 to 14.

Figure 6.

Effects of CPT-cGMP on fully opened channels. (A) Representative traces showing the responses of αβ_S520Cγ to CPT-cGMP (0.2 mM) before and after the addition of MTSET (1 mM). (B) Activation of ENaC by CPT-cGMP. The current ratios are 3.1 ± 0.1 before MTSET and 1.1 ± 0.03 after MTSET, respectively. ***P < 0.001. n = 11.

Figure 7.

Analysis of cell cGMP contents by ELISA. (A) Average cell cGMP contents in instilled lungs (left), H441 cells (middle), and oocytes (right). n = 6 lungs, 10 H441 monolayers, and 100 oocytes. **P < 0.01. (B) Divergent regulation of ENaC by CPT-cGMP in oocytes (left) and epithelial cells (right). Heterologous ENaC channels are not regulated by cGMP/PKG and cAMP/PKA pathways. CPT-cGMP therefore does not regulate cloned human ENaC in oocytes via the cGMP/PKG signaling pathway and only serves as an external ligand. This compound stimulates native ENaC function by acting as an external ligand and activating internal PKG in sequence. cGMP is anticipated to accumulate around ENaC as an autocrine.