NBCe1 and NBCn1 are expressed in human submandibular gland (hSMG) and HSG cells.

<p>(A) NBCe1 and NBCn1 mRNA transcripts in hSMG and HSG cells. Aquaporin 5 (AQP5) was used as a marker for acinar cells. (B and C) hSMG tissue sections were stained with NBCe1, NBCn1, and AQP5 antibodies. (Bar = 50 μm). AQP5 was used as a marker for acinar cells. White and yellow arrows indicate acinar cells and duct cells, respectively. NBCe1-B is expressed in human submandibular gland (hSMG) duct cells, whereas NBCn1 is expressed in acinar (white arrow) and duct cells (yellow arrow). (D and E) HSG cells were stained with antibodies for NBCe1 and NBCn1. (Bar = 20 μm).</p

pH_i recovery of HSG cells is inhibited by PP2.

<p>(A and B) The pH_i of HSG cells in HBS was obtained in the absence or presence of 5, 10, or 25 μM EIPA and the results were summarized (C-F) pH_i measurements were performed using HSG cells in BBS, and the effects of pre-treatment with 5 μM PP2 for 20 min were evaluated (grey trace). (G) Summary of pH_i recovery rates in HSG cells. The data are presented as the mean ± S.E. (error bars) (*, P < 0.05; ***, P < 0.001)</p

PP2 inhibits tyrosine phosphorylation and translocation of NBCe1-B, but NBCn1, in HSG cells.

<p>(A and B) Cell lysates were subjected to immunoprecipitation with NBCe1 and NBCn1 antibodies and evaluated by Western blotting with a phosphotyrosine antibody. The cells were pre-incubated in 20 mM of NH₄Cl for 2 mins, in 5 μM of PP2 for 20 mins, and in 50 μM of CCh for 5 mins. The input control comprised 5% of the lysates. (C and D) Phosphorylated NBCe1 and NBCn1 were quantified based on protein band intensities. The data are shown as the mean ± S.E. (error bars) (n = 4; *, P < 0.05). (E) Locations of NBCe1 and NBCn1 in response of ammonium pulse in the presence or absence of PP2 were confirmed using immunocytochemistry. (Bar = 20 μm).</p

Transfected NBCe1-B is affected by PP2.

<p>(A and B) Flag-NBCe1-B and NBCn1 were transfected into HSG cells and overexpression was confirmed by immunofluorescence assay. (C and D) pH_i recovery rates were recorded in HSG cells overexpressing NBCe1-B or NBCn1. Horizontal bars indicate all applications. (E and F) Graphical summary of pH_i recovery rates. The data are presented as the mean ± S.E. (error bars) (*, P < 0.05; ***, P < 0.001).</p

Role of Sodium Bicarbonate Cotransporters in Intracellular pH Regulation and Their Regulatory Mechanisms in Human Submandibular Glands

<div><p>Sodium bicarbonate cotransporters (NBCs) are involved in the pH regulation of salivary glands. However, the roles and regulatory mechanisms among different NBC isotypes have not been rigorously evaluated. We investigated the roles of two different types of NBCs, electroneutral (NBCn1) and electrogenic NBC (NBCe1), with respect to pH regulation and regulatory mechanisms using human submandibular glands (hSMGs) and HSG cells. Intracellular pH (pH_i) was measured and the pH_i recovery rate from cell acidification induced by an NH₄Cl pulse was recorded. Subcellular localization and protein phosphorylation were determined using immunohistochemistry and co-immunoprecipitation techniques. We determined that NBCn1 is expressed on the basolateral side of acinar cells and the apical side of duct cells, while NBCe1 is exclusively expressed on the apical membrane of duct cells. The pH_i recovery rate in hSMG acinar cells, which only express NBCn1, was not affected by pre-incubation with 5 μM PP2, an Src tyrosine kinase inhibitor. However, in HSG cells, which express both NBCe1 and NBCn1, the pH_i recovery rate was inhibited by PP2. The apparent difference in regulatory mechanisms for NBCn1 and NBCe1 was evaluated by artificial overexpression of NBCn1 or NBCe1 in HSG cells, which revealed that the pH_i recovery rate was only inhibited by PP2 in cells overexpressing NBCe1. Furthermore, only NBCe1 was significantly phosphorylated and translocated by NH₄Cl, which was inhibited by PP2. Our results suggest that both NBCn1 and NBCe1 play a role in pH_i regulation in hSMG acinar cells, and also that Src kinase does not regulate the activity of NBCn1.</p></div

Src tyrosine kinase does not affect pH_i recovery of hSMG acinar cells.

<p>(A and B) The intracellular pH recovery patterns of hSMG acinar cells in the absence or presence of several concentrations of EIPA in HEPES-buffered solution (HBS) were measured and the pH_i recovery rates were summarized. (C-F) The pH_i recovery patterns of hSMG acinar cells following an NH₄⁺-pulse (blank bar) were recorded in a bicarbonate-buffered bath solution (BBS). The cells were pretreated for 20 min with 5 μM PP2, a Src tyrosine kinase inhibitor (grey trace) or incubated in normal BBS (black trace). The effects of treatment with EIPA and DIDS are shown using horizontal bars. (G) Summary of pH_i recovery rates. The data are presented as the mean ± S.E.</p

Schematic model of NBCe1-B and NBCn1 regulation by Src kinase.

<p>NBCe1-B and NBCn1 mediate intracellular pH in an HSG cell line and human submandibular glands, especially on the apical side of duct cells. NBCe1-B is phosphorylated by Src kinase and translocates to the plasma membrane, whereas the NBCn1 is not regulated by Src kinase. The effect of Src kinase is inhibited by PP2. Arrows indicate activation and bars indicate inhibition.</p

List of DNA primers sequences designed for RT-PCR.

<p>List of DNA primers sequences designed for RT-PCR.</p

Additional file 1 of RIPK3 activation induces TRIM28 derepression in cancer cells and enhances the anti-tumor microenvironment

Additional file 1