Attenuated Amiloride-Sensitive Current and Augmented Calcium-Activated Chloride Current in Marsh Rice Rat (Oryzomys palustris) Airways

Abstract

Prolonged heat and sea salt aerosols pose a challenge for the mammalian airway, placing the protective airway surface liquid (ASL) at risk for desiccation. Thus, mammals inhabiting salt marshes might have acquired adaptations for ASL regulation. We studied the airways of the rice rat, a rodent that inhabits salt marshes. We discovered negligible Na⁺ transport through the epithelial sodium channel (ENaC). In contrast, carbachol induced a large Cl^- secretory current that was blocked by the calcium-activated chloride channel (CaCC) inhibitor CaCCinhi-A01. Decreased mRNA expression of α, β, and γ ENaC, and increased mRNA expression of the CaCC transmembrane member 16A, distinguished the rice rat airway. Rice rat airway cultures also secreted fluid in response to carbachol and displayed an exaggerated expansion of the ASL volume when challenged with 3.5% NaCl. These data suggest that the rice rat airway might possess unique ion transport adaptations to facilitate survival in the salt marsh environment.

Keywords: Bio-Electrochemistry: Physiology; Molecular Physiology.

Graphical abstract

Figure 1

Tight Junctions and Cilia in Rice Rat Cultures (A–L) En face images of tracheal cultures of mouse (A), piglet (B), and rice rat (C) showing expression of the cilia protein acetylated alpha tubulin. En face images of tracheal cultures of mouse (D), piglet (E), and rice rat (F) showing expression of the tight junction protein ZO-1. En face images of tracheal cultures of mouse (G), piglet (H), and rice rat (I) incubated with only the goat anti-mouse 488 secondary antibody (secondary used for the detection of acetylated tubulin). En face images of tracheal cultures of mouse (J), piglet (K), and rice rat (L) incubated with only goat anti-rabbit 488 secondary antibody; this secondary was used for the detection of ZO-1. ZO-1, zonula occludens protein 1. Hoechst was used to detect nuclei. For all panels, scale bars, 50 μm. Antibody detection of ZO-1 and acetylated alpha tubulin was performed on two separate occasions for each species. White arrows show example of specific staining.

Figure 2

Primary Tracheal Cultures of Rice Rats Have Limited Basal and Amiloride-Sensitive Current, but Large Carbachol-Mediated Cl⁻ Secretion (A–H) Basal short circuit current (Isc) measurements in primary cultures of rice rat and piglet (A). Averaged delta (Δ) Isc measurements shown for 100 μM apical amiloride (AMIL) (B), 100 μM basolateral carbachol (CCh) (peak and sustained responses in C and D, respectively), 30 μM apical CaCCinhi-A01for inhibition of peak (E) and sustained (F) CCh-mediated current, apical 10 μM forskolin and 100 μM IBMX (F&I) (G), and 100 μM apical GLYH-101 (H). (I–K) Representative short circuit current (Isc) trace in mouse (I), piglet (J), and rice rat (K) airway cultures. Arrows indicate addition of drugs (as described in Methods). CCh, carbachol; F&I, forskolin and IBMX. For all panels, n = 4 cultures from mice (2 female cultures and 2 male cultures representing 6 female and 6 male subjects); n = 7 cultures from the rice rat (4 female cultures and 3 male cultures representing 8 female and 6 male subjects); n = 9 porcine cultures (5 female and 4 male representing 5 individual female and 4 individual male subjects). ∗p < 0.05 compared with mouse; ^#p < 0.05 compared with pig. All data are shown as mean ± SEM.

Figure 3

Rice Rat Tracheal Cultures have Decreased ENaC Expression and Increased TMEM16A Expression Compared with Mice (A–K) mRNA expression of α ENaC (A), β ENaC (B), and γ ENaC (C) in mouse and rice rat airway cultures. mRNA expression of α ENaC (D), β ENaC (E), and γ ENaC (F) in rice rat airway cultures treated with dexamethasone (DEX) and aldosterone (ALDO). mRNA expression of NR3C1 (G), NR3C2 (H), TMEM16A (I), CFTR (J), and CHRM3 (K). ENaC, epithelial sodium channel; CFTR, cystic fibrosis transmembrane conductance regulator; NR3C1, glucocorticoid receptor; NR3C2, mineralocorticoid receptor; TMEM16, transmembrane member 16A; CHRM3, muscarinic acetylcholine receptor 3. For (A–C) and (G–K), n = 4 cultures from mice (2 female cultures and 2 male cultures representing 6 female and 6 male subjects); n = 4 cultures from the rice rat (2 female cultures and 2 male cultures representing 6 female and 6 male subjects). For (D–F), n = 3 cultures from the rice rat (2 female cultures and 1 male culture representing 6 female and 3 male subjects). *p < 0.05 compared with mouse. All data are shown as mean ± SEM.

Figure 4

Rice Rat Primary Cultures Secrete Fluid in Response to Carbachol (A–C) Apical fluid secretion in response to 100 μM basolateral carbachol in mouse (A), pig (B), and rice rat (C) airway cultures. (D) Fluid secretion responses normalized to baseline values and expressed as a percent for each species. For all panels, n = 4 cultures from mice (2 female cultures and 2 male cultures representing 6 female and 6 male subjects); n = 6 cultures from the rice rat (3 female cultures and 3 male cultures representing 6 female and 6 male subjects); n = 6 porcine cultures (3 female and 3 male representing 3 individual female and 3 individual male subjects). ∗p < 0.05 compared with baseline. All data are shown as mean ± SEM.

Figure 5

Rice Rat Primary Cultures Display an Exaggerated Fluid Expansion to Hypertonic Saline (A–C) Surface fluid expansion in response to apical 3.5% NaCl increased in in mouse (A), pig (B), and rice rat (C) airway cultures. (D) Fluid expansion responses normalized to baseline values and expressed as a percent for each species. For all panels, n = 4 cultures from mice (2 female cultures and 2 male cultures representing 6 female and 6 male subjects); n = 6 cultures from the rice rat (3 female cultures and 3 male cultures representing 6 female and 6 male subjects); n = 6 porcine cultures (3 female and 3 male representing 3 individual female and 3 individual male subjects). For (B–C) ∗p < 0.05 compared with baseline. For (D) ∗p < 0.05 compared with mouse; #p < 0.05 compared with pig. All data are shown as mean ± SEM.