The Na+/HCO3− Cotransporter (Nbce1, Slc4a4) is Enriched in Interstitial Cells of Cajal Responsible for Generating Electrical Slow Wave Activity in the Mouse Gastrointestinal Tract
Authors: Colmenares Aguilar Maria G. , Zhao Wenchang , Mazzone Amelia , Strege Peter R. , Eisenman Seth T. , Sha Lei , Shull Gary E. , Romero Michael F. , Farrugia Gianrico , Gibbons Simon J. 
Published: June 2021

Electrical slow wave activity, generated by interstitial cells of Cajal (ICC), is essential for efficient gastrointestinal (GI) transit. Electrophysiology of slow waves has been extensively studied; inclusion of bicarbonate (HCO3−) in extracellular solutions is required for recording normal slow wave activity. HCO3− transport contributes to regulation of intracellular pH, which has multiple consequences for normal cellular function. Previous transcriptional profiling found that the Na+/ HCO3− cotransporter (NBCe1/Slc4a4) is enriched in pacemaker myenteric ICC (ICC‐MY) compared to non pacemaker ICC in the deep muscular plexus (ICC‐DMP) of the small intestine. Our aim was to determine the distribution and identity of Slc4a4/NBCe1 variants in ICC in the GI tract.

NBCe1 immunoreactivity (IR) was detected by fluorescence labeling of parafomaldehyde‐fixed cross sections and whole mounts of adult mouse small intestine, colon and stomach using primary antibodies directed against a 329 amino acid sequence in the N terminus of the NBCe1 protein. Labeling of Kit receptor tyrosine kinase with a goat anti‐Kit polyclonal antibody was used to detect ICC. Slc4a4 knockout (KO) mice were bred from heterozygous founders. Slc4a4fl/fl mice were bred with ETV1CreERT2/− mice to allow targeted, conditional KO of Slc4a4 in ICC in response to tamoxifen treatment (130 mg/Kg). mRNA transcripts for Slc4a4 in Kit expressing cells were isolated from Kit‐ribotag mice, which were generated by breeding KitcreERT2 with Rpl22tm1.1Psam mice. The abundance of specific Slc4a4 transcripts was measured by quantitative real time PCR (qRT‐PCR). Green fluorescent protein (GFP)‐labeled ICC were dissociated from the small intestine of neonatal KitcopGFP mice and RNA expression levels were determined using the Single Cell‐to‐CT™ qRT‐PCR Kit from Ambion.

NBCe1‐IR was absent in all cells in Slc4a4‐KO tissues but co‐localized with Kit in ICC‐MY, but not in ICC‐DMP, in small intestine of wild type mice. In the proximal and distal colon, pacemaker submucosal ICC (ICC‐SMP) but not intramuscular ICC (ICC‐IM) or ICC‐MY showed NBCe1‐IR. In the corpus and antrum of the stomach NBCe1‐IR was detected in ICC‐MY but not in ICC‐IM. NBCe1‐IR did not co‐localize with Kit in the gastric fundus. mRNA for variant C, but not variants A or B of Slc4a4, was detected in small intestinal ICC‐MY. Reduced NBCe1‐IR was restricted to small intestinal ICC‐MY of Slc4a4 conditional KO mice.

NBCe1 is enriched in subtypes of ICC responsible for pacemaker activity in all regions of the GI tract indicating a role for this transporter in slow wave generation. The presence of the C variant of Slc4a4 in ICC‐MY suggests dynamic regulation of NBCe1 activity in ICC by the IP3R binding protein released with inositol 1, 4, 5‐trisphosphate (IRBIT).

Support or Funding Information

Supported by NIH R01 DK57061.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.