cAMP-dependent and cholinergic regulation of the electrogenic intestinal/pancreatic Na+/HCO3- cotransporter pNBC1 in human embryonic kidney (HEK293) cells

<p>Abstract</p> <p>Background</p> <p>The renal (kNBC1) and intestinal (pNBC1) electrogenic Na⁺/HCO₃^-cotransporter variants differ in their primary structure, transport direction, and response to secretagogues. Previous studies have suggested that regulatory differences between the two subtypes can be partially explained by unique consensus phosphorylation sites included in the pNBC1, but not the kNBC1 sequence. After having shown activation of NBC by carbachol and forskolin in murine colon, we now investigated these pathways in HEK293 cells transiently expressing a GFP-tagged pNBC1 construct. </p> <p>Results</p> <p>Na⁺- and HCO₃^--dependent pH_irecovery from an acid load (measured with BCECF) was enhanced by 5-fold in GFP-positive cells compared to the control cells in the presence of CO₂/HCO₃^-. Forskolin (10^-5M) had no effect in untransfected cells, but inhibited the pH_irecovery in cells expressing pNBC1 by 62%. After preincubation with carbachol (10^-4M), the pH_irecovery was enhanced to the same degree both in transfected and untransfected cells, indicating activation of endogenous alkalizing ion transporters. Acid-activated Na⁺/HCO₃^-cotransport via pNBC1 expressed in renal cells is thus inhibited by cAMP and not affected by cholinergic stimulation, as opposed to the findings in native intestinal tissue. </p> <p>Conclusion</p> <p>Regulation of pNBC1 by secretagogues appears to be not solely dependent on its primary structure, but also on properties of the cell type in which it is expressed.</p

An unusually powerful mode of low-frequency sound interference due to defective hair bundles of the auditory outer hair cells

International audienceA detrimental perceptive consequence of damaged auditory sen-sory hair cells consists in a pronounced masking effect exerted by low-frequency sounds, thought to occur when auditory threshold elevation substantially exceeds 40 dB. Here, we identified the submembrane scaffold protein Nherf1 as a hair-bundle component of the differentiating outer hair cells (OHCs). Nherf1 −/− mice dis-played OHC hair-bundle shape anomalies in the mid and basal co-chlea, normally tuned to mid-and high-frequency tones, and mild (22–35 dB) hearing-threshold elevations restricted to midhigh sound frequencies. This mild decrease in hearing sensitivity was, however, discordant with almost nonresponding OHCs at the co-chlear base as assessed by distortion-product otoacoustic emissions and cochlear microphonic potentials. Moreover, unlike wild-type mice, responses of Nherf1 −/− mice to high-frequency (20–40 kHz

Crosstalk Between Innate and T Cell Adaptive Immunity With(in) the Muscle

Growing evidence demonstrates a continuous interaction between the immune system and the skeletal muscle in inflammatory diseases of different pathogenetic origins, in dystrophic conditions such as Duchenne Muscular Dystrophy as well as during normal muscle regeneration. Although one component of the innate immunity, the macrophage, has been extensively studied both in disease conditions and during cell or gene therapy strategies aiming at restoring muscular functions, much less is known about dendritic cells and their primary immunological targets, the T lymphocytes. This review will focus on the dendritic cells and T lymphocytes (including effector and regulatory T-cells), emphasizing the potential cross talk between these cell types and their influence on the structure and function of skeletal muscle

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