cGMP stimulates sodium and chloride currents in rat tracheal airway epithelia

Abstract

To test the hypothesis that guanosine 3',5'-cyclic monophosphate (cGMP) regulates ion transport in airway epithelial cells, we measured short-circuit current (I(sc)) and (22)Na+ fluxes in primary cultured rat tracheal epithelial cells. In Cl- -containing Ringer solution, I(sc) was increased by approximately 17 microA/cm2 after application of 1 mM 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP), whereas, in Cl- -free solutions, the Na+ -mediated component was approximately 5 microA/cm2, suggesting a cGMP stimulation of Cl-secretory current and a smaller Na+ absorptive current. Inward and net mucosal-to-serosal (22)Na+ flux was doubled in the presence of 2 mM 8-BrcGMP. To determine whether nucleotide-gated channels play a role in this transepithelial Na+ absorption, blockers of nucleotide-gated cation channels were used to inhibit I(sc). The cGMP-stimulated Na+-mediated I(sc) was blocked by as little as 500 nM dichlorobenzamil or 50 microM L-cis-diltiazem, which are known blockers for cyclic nucleotide-gated cation channels. These agents also blocked the basal (non-cGMP-stimulated) current when measured in the presence of 10 microM amiloride, which blocks current through 5-pS amiloride-sensitive Na+ channels. To document whether the distribution of nucleotide-gated nonselective cation channels was consistent with a role in airway epithelial transport, in situ hybridization was performed. In situ hybridization of mRNA encoding for nucleotide-gated cation channels was found in epithelial cell layers of rat trachea, bronchi, bronchioles, and alveolar cells but not in smooth muscle layers or tracheal cartilage. Reverse transcriptase-polymerase chain reaction, restriction enzyme analysis, and sequencing of the cDNA transcribed from mRNA of whole lung and tracheal epithelial cells indicate that a channel highly homologous to the retinal nucleotide-gated nonselective cation channel (CNG1) is present. Thus these data, along with evidence supporting the existence of signal transduction pathways elevating intracellular levels of cGMP, indicate that cGMP regulates transepithelial ion transport in lung epithelial tissues.

8-(4-Chlorophenylthio)-Guanosine-3′,5′-Cyclic Monophosphate-Na Stimulates Human Alveolar Fluid Clearance by Releasing External Na+ Self-Inhibition of Epithelial Na+ Channels

“…cGMP did not alter the short-circuit current (Isc) across rat ATII monolayers (39), supporting a previous study using dome formation as a parameter for active salt and water transport (40). In contrast, cGMP, as well as NO, increased Isc and 22 Na influx in tracheal and distal lung epithelia (41,42). In addition, regulation of Na 1 channel activity in A549 and rat ATII cells, as revealed by patch clamp studies, is not consistent.…”

Regulation of the Epithelial Sodium Channel by Serine Proteases in Human Airways

“…In addition, while ENaC is thought to be the dominant amiloride-sensitive pathway in airway epithelia, we have not ruled out a serine protease effect on other amiloride-sensitive, electrogenic pathways (e.g. cyclic nucleotide gated channels (26)), which could contribute to our experimental results.…”

Lung Epithelial Fluid Transport and the Resolution of Pulmonary Edema

“…There is some limited evidence that the amilorideinsensitive fraction of 8-Br-cGMP stimulated I sc and sodium uptake in rat tracheal epithelia was inhibited by dichlorobenzamil or L-cis-diltiazem, both inhibitors of cyclic nucleotide-gated cation channels (CNG) (322). In subsequent studies, dichlorobenzamil inhibited a significant fraction of lung fluid absorption in sheep (170), a finding that supported the hypothesis that CNG channels could play a role in fluid absorption from the distal airspaces.…”