Abstract
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 pHi 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.
Highlights
Electrogenic Na+/HCO32 cotransporter NBCe1 exists in 2 basolaterally localized variants (SLC4 family members NBCe1-A and NBCe1-B) with major differences in their transport direction, stoichiometry, expression pattern, and regulation [1]
Basolateral HCO32 uptake in the intestinal epithelium by specific transporters is believed to be essential for intracellular pH regulation and further functions of HCO32, and rate-limiting for transepithelial HCO32 transport, the role of Protein kinase C (PKC) in the regulation of intestinal HCO32 uptake has not been studied in detail
In this study designed to investigate the role of PKC during secretagogue-associated activation of Na+/HCO32 cotransporter (NBC), we first asked how PKC isoforms are subcellularly localized in murine colon, and whether PKC activation by forskolin and carbachol occurs in isolated crypts
Summary
Electrogenic Na+/HCO32 cotransporter NBCe1 exists in 2 basolaterally localized variants (SLC4 family members NBCe1-A and NBCe1-B) with major differences in their transport direction, stoichiometry, expression pattern, and regulation [1]. The importance of physiologically relevant regulatory pathways has been investigated, and these studies have revealed a differential regulation of NBCe1-A and NBCe1-B by cholinergic and cAMP-dependent stimulation [2,5,6,7,8]. These differences have been attributed to the regulatory properties of the respective variant arising from the primary structure [9,10,11], and to celltype specific factors [5,10], underlining the importance of data derived from experiments using native tissue rather than heterologous expression systems. Basolateral HCO32 uptake in the intestinal epithelium by specific transporters is believed to be essential for intracellular pH regulation and further functions of HCO32, and rate-limiting for transepithelial HCO32 transport, the role of PKC in the regulation of intestinal HCO32 uptake has not been studied in detail
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