Capsaicin induces NKCC1 internalization and inhibits chloride secretion in colonic epithelial cells independently of TRPV1

Abstract

Colonic chloride secretion is regulated via the neurohormonal and immune systems. Exogenous chemicals (e.g., butyrate, propionate) can affect chloride secretion. Capsaicin, the pungent ingredient of the chili peppers, exerts various effects on gastrointestinal function. Capsaicin is known to activate the transient receptor potential vanilloid type 1 (TRPV1), expressed in the mesenteric nervous system. Recent studies have also demonstrated its presence in epithelial cells but its role remains uncertain. Because capsaicin has been reported to inhibit colonic chloride secretion, we tested whether this effect of capsaicin could occur by direct action on epithelial cells. In mouse colon and model T84 human colonic epithelial cells, we found that capsaicin inhibited forskolin-dependent short-circuit current (FSK-I(sc)). Using PCR and Western blot, we demonstrated the presence of TRPV1 in colonic epithelial cells. In T84 cells, TRPV1 localized at the basolateral membrane and in vesicular compartments. In permeabilized monolayers, capsaicin activated apical chloride conductance, had no effect on basolateral potassium conductance, but induced NKCC1 internalization demonstrated by immunocytochemistry and basolateral surface biotinylation. AMG-9810, a potent inhibitor of TRPV1, did not prevent the inhibition of the FSK-I(sc) by capsaicin. Neither resiniferatoxin nor N-oleoyldopamine, two selective agonists of TRPV1, blocked the FSK-I(sc). Conversely capsaicin, resiniferatoxin, and N-oleoyldopamine raised intracellular calcium ([Ca(2+)](i)) in T84 cells and AMG-9810 blocked the rise in [Ca(2+)](i) induced by capsaicin and resiniferatoxin suggesting the presence of a functional TRPV1 channel. We conclude that capsaicin inhibits chloride secretion in part by causing NKCC1 internalization, but by a mechanism that appears to be independent of TRPV1.

A patient with multisystem dysfunction carries a truncation mutation in human SLC12A2, the gene encoding the Na-K-2Cl cotransporter, NKCC1

“…One cation-chloride cotransporter that has eluded the list of cotransporters with human mutations is the ubiquitous Na-K-2Cl cotransporter-1 or NKCC1. NKCC1, like its family of transporters, plays a role in epithelial ion transport (O'Mahony et al 2008; Bouyer et al 2013; Kidokoro et al 2014; Wei et al 2015), modulates inhibitory synaptic transmission (Sung et al 2000; Dzhala et al 2005), and maintains and regulates cell volume (Wu et al 1998; Bush et al 2010; Mathieu et al 2015). Accordingly, the knockout mouse exhibits multiple phenotypes that include sensorineural deafness (Delpire et al 1999; Dixon et al 1999; Flagella et al 1999), male infertility (Pace et al 2000), intestinal transit deficiency (Flagella et al 1999; Wouters et al 2006; Bradford et al 2016), deficit in saliva secretion (Evans et al 2000), and a pain perception phenotype (Sung et al 2000; Laird et al 2004; Granados-Soto et al 2005).…”

Impact of Hybrid and Complex N-Glycans on Cell Surface Targeting of the Endogenous Chloride CotransporterSlc12a2

“…With some exceptions, such as tubular cells of the thick ascending limb of Henle's loop (TALH) [ 8 ] or glucagon-secreting cells of the endocrine pancreas [ 9 , 10 ], NKCC1 has been found in all mammalian cells examined so far at the protein or functional level [ 11 ]. In particular, NKCC1 localizes in the basolateral side of most epithelial cells [ 12 – 15 ] and polarized cell lines [ 16 – 18 ]. In nonpolarized cells including primary astrocytes [ 19 ] or insulin-secreting cells [ 10 , 20 ], NKCC1 is found abundantly in cytoplasmic compartments.…”

AMPK mediates inhibition of electrolyte transport and NKCC1 activity by reactive oxygen species

“…NKCC1 biotinylation assay. NKCC1 surface expression was determined as previously described (7). Briefly, T84 monolayers were treated with 500 M H2O2 with or without 30 min of pretreatment with compound C (50 M), metformin (5 mM) alone, or capsaicin (20 M) alone.…”

“…H 2 O 2 -activated AMPK does not promote internalization of membrane-bound NKCC1. NKCC1 has also been shown to be regulated via internalization of membrane-bound NKCC1 in colonic epithelial cells (7). Therefore, we employed a surface biotinylation assay to determine if surface expression of NKCC1 is altered and if NKCC1 internalization is AMPKdependent.…”