Short-Term Regulation of Murine Colonic NBCe1-B (Electrogenic Na⁺/HCO₃⁻ Cotransporter) Membrane Expression and Activity by Protein Kinase C

<div><p>The colonic mucosa actively secretes HCO₃⁻, and several lines of evidence point to an important role of Na⁺/HCO₃⁻ cotransport (NBC) as a basolateral HCO₃⁻ import pathway. We could recently demonstrate that the predominant NBC isoform in murine colonic crypts is electrogenic NBCe1-B, and that secretagogues cause NBCe1 exocytosis, which likely represents a component of NBC activation. Since protein kinase C (PKC) plays a key role in the regulation of ion transport by trafficking events, we asked whether it is also involved in the observed NBC activity increase. Crypts were isolated from murine proximal colon to assess PKC activation as well as NBC function and membrane abundance using fluorometric pH_i measurements and cell surface biotinylation, respectively. PKC isoform translocation and phosphorylation occurred in response to PMA-, as well as secretagogue stimulation. The conventional and novel PKC inhibitors Gö6976 or Gö6850 did not alter NBC function or surface expression by themselves, but stimulation with forskolin (10⁻⁵ M) or carbachol (10⁻⁴ M) in their presence led to a significant decrease in NBC-mediated proton flux, and biotinylated NBCe1. Our data thus indicate that secretagogues lead to PKC translocation and phosphorylation in murine colonic crypts, and that PKC is necessary for the increase in NBC transport rate and membrane abundance caused by cholinergic and cAMP-dependent stimuli.</p></div

Immunohistochemical staining of NBCe1 and PKC isoforms in murine colonic tissue sections.

<p>NBCe1 (A) is expressed in the basolateral membrane of colonic crypts. Cytoplasmatic expression was observed for PKCα (C), δ (E) and ε (G) isoforms, with an apparent slight accumulation of the signal in the vicinity of the cell membrane for PKC α and PKC ε. Control experiments without the secondary antibody (NBCe1, B) and blocking peptides (PKC isoforms, D/F/H) showed no specific signal. Size of the scale bar is 50 μm.</p

NBCe1 membrane expression during secretagogue stimulation and PKC inhibition with Gö6850 or Gö6976.

<p>In cell surface biotinylation experiments, neither Gö6850 (A; 5 μM) nor Gö6976 (B; 5 μM; continuous incubation for 20 min [+] vs. vehicle [−]) caused changes in NBCe1 surface expression. When forskolin (10⁻⁵ M) or carbachol (10⁻⁴ M) were added after 10 min, NBCe1 surface expression significantly decreased (40 and 59% for Gö6850, and 8 and 31% for Gö6976, respectively; n = 5–7 preparations from separate mice in each group, *:p<0.05, ANOVA for correlated samples followed by Tukey's HSD).</p

PKC phosphorylation in response to PMA and secretagogues.

<p>Phosphorylation was assessed in crypt lysates using the respective antibody recognizing the phosphorylated isoform. All compounds caused an increased in phosphorylated PKC-α (A) and PKC-ε (B; preincubation 10 min. 37°C; **:p<0.05, *:p<0.05 vs. unstimulated). When Gö6850 (5 μM) was added 10 min prior to stimulation, the effect of stimulation on p-PKC-α was completely reversed (A; #:p<0.01, *:p<0.05). PKC-ε phosphorylation, however, was only inhibited in the case of PMA (100 nM), but not secretagogues (B; #:p<0.01, *:p<0.05; n =  6 preparations from separate mice in each group, ANOVA for correlated samples followed by Tukey's HSD).</p

PKC translocation in response to PMA and secretagogues.

<p>PKC membrane expression as assessed by surface biotinylation and probing with a general PKC antibody significantly increased after exposure to PMA (100 nM), forskolin (10⁻⁵ M), or carbachol (10⁻⁴ M, 10 min each), which was paralleled by a decrease in cytosolic PKC expression [ODI: optical density integrated; *:p<0.05 vs. unstimulated, n =  6 preparations from separate mice in each group, ANOVA for correlated samples followed by Tukey's HSD (Tukey's honestly significant difference test), values expressed as % of the unstimulated control for cytosol and membrane, respectively].</p

pH-microfluorometrical determination of NBC activity in the presence of PKC inhibitors with and without forskolin stimulation.

<p>NBC transport rates were determined as the Na⁺- and CO₂/HCO₃⁻-dependent, DMA-insensitive proton flux rates during pH_i recovery from an acid load. A: Average pH_i trace (n = 5) illustrating the pH_i recovery protocol, where crypts were acidified with an NH₄ “prepulse” to a pH of 6.4±0.2 in Na⁺-free buffer (TMA-Cl) and let to recover after Na⁺ re-addition in the presence of 700 μM DMA to block all Na⁺/H⁺ exchanger isoforms. In the absence of CO₂/HCO₃⁻, no significant pH_i recovery was observed, but in its presence, there was a steady pH_i increase representing Na⁺/HCO₃⁻ cotransport (NBC; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092275#pone.0092275-Bachmann1" target="_blank">[2]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092275#pone.0092275-Bachmann3" target="_blank">[8]</a>). B: Left side: Incubation with Gö6976 (5 μM, 10 min prior to stimulation, light grey bars) or Gö6850 (5 μM, 10 min prior to stimulation, dark grey bars) did not alter the control proton flux rates (solid bars; p = n.s.). Right side: Stimulation with forskolin led to the previously observed significant stimulation of NBC (10.2±0.8 vs. 5.3±0.4 mM/min; *:p<0.05). Preincubation with either of the PKC inhibitors completely reversed this effect (n = 5–7 experiments from separate mice, p = n.s., ANOVA for independent samples followed by Tukey's HSD).</p

Additional file 1 of Highly selective whole-cell 25-hydroxyvitamin D3 synthesis using molybdenum-dependent C25-steroid dehydrogenase and cyclodextrin recycling

Supplementary Material