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Sphingomyelinase decreases transepithelial anion secretion in airway epithelial cells in part by inhibiting CFTR-mediated apical conductance.

ABSTRACT: The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel whose dysfunction causes cystic fibrosis (CF). The loss of CFTR function in pulmonary epithelial cells causes surface dehydration, mucus build-up, inflammation, and bacterial infections that lead to lung failure. Little has been done to evaluate the effects of lipid perturbation on CFTR activity, despite CFTR residing in the plasma membrane. This work focuses on the acute effects of sphingomyelinase (SMase), a bacterial virulence factor secreted by CF relevant airway bacteria which degrades sphingomyelin into ceramide and phosphocholine, on the electrical circuitry of pulmonary epithelial monolayers. We report that basolateral SMase decreases CFTR-mediated transepithelial anion secretion in both primary bronchial and tracheal epithelial cells from explant tissue, with current CFTR modulators unable to rescue this effect. Focusing on primary cells, we took a holistic ion homeostasis approach to determine a cause for reduced anion secretion following SMase treatment. Using impedance analysis, we determined that basolateral SMase inhibits apical and basolateral conductance in non-CF primary cells without affecting paracellular permeability. In CF primary airway cells, correction with clinically relevant CFTR modulators did not prevent SMase-mediated inhibition of CFTR currents. Furthermore, SMase was found to inhibit only apical conductance in these cells. Future work should determine the mechanism for SMase-mediated inhibition of CFTR currents, and further explore the clinical relevance of SMase and sphingolipid imbalances.

SUBMITTER: Cottrill KA

PROVIDER: S-EPMC8358481 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Human bronchial epithelial (HBE) cells exhibit constitutive anion secretion that is absent in cells from cystic fibrosis (CF) patients. The identity of this conductance is unknown, but SLC26A9, a member of the SLC26 family of CF transmembrane conductance regulator (CFTR)-interacting transporters, is found in the human airway and exhibits chloride channel behavior. We sought differences in the properties of SLC26A9 and CFTR expressed in HEK 293 (HEK) cells as a fingerprint to identify HBE apical anion conductances. HEK cells expressing SLC26A9 displayed a constitutive chloride current that was inhibited by the CFTR blocker GlyH-101 (71 +/- 4%, 50 microM) and exhibited a near-linear current-voltage (I-V) relation during block, while GlyH-101-inhibited wild-type (wt)CFTR exhibited a strong inward-rectified (IR) I-V relation. We tested polarized HBE cells endogenously expressing either wt or DeltaF508-CFTR for similar activity. After electrical isolation of the apical membrane using basolateral alpha-toxin permeabilization, wtCFTR monolayers displayed constitutive chloride currents that were inhibited by GlyH-101 (68 +/- 6%) while maintaining a near-linear I-V relation. In the absence of blocker, the addition of forskolin stimulated a current increase having a linear I-V; GlyH-101 blocked 69 +/- 7% of the current and shifted the I-V relation IR, consistent with CFTR activation. HEK cells coexpressing SLC26A9 and wtCFTR displayed similar properties, as well as forskolin-stimulated currents that exceeded the sum of those in cells separately expressing SLC26A9 or wtCFTR, and an I-V relation during GlyH-101 inhibition that was moderately IR, indicating that SLC26A9 contributed to the stimulated current. HBE cells from CF patients expressed SLC26A9 mRNA, but no constitutive chloride currents. HEK cells coexpressing SLC26A9 with DeltaF508-CFTR also failed to exhibit SLC26A9 current. We conclude that SLC26A9 functions as an anion conductance in the apical membranes of HBE cells, it contributes to transepithelial chloride currents under basal and cAMP/protein kinase A-stimulated conditions, and its activity in HBE cells requires functional CFTR.

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Sphingomyelinase decreases transepithelial anion secretion in airway epithelial cells in part by inhibiting CFTR-mediated apical conductance.

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