Endotoxin differentially modulates the basolateral and apical sodium/proton exchangers (NHE) in enterocytes.
BACKGROUND
Maintenance of enterocyte activity during extracellular acidosis requires functional sodium/proton exchangers (NHE), which are present at both basolateral and apical surfaces. Necrotizing enterocolitis is characterized by systemic hypoperfusion, metabolic acidosis, and the apical to basolateral translocation of endotoxin (lipopolysaccharide [LPS]). We hypothesized that LPS differentially impairs NHE activity at the basolateral or apical domains of enterocytes, leading to cellular acidification, and explored the mechanisms involved.


METHODS
Experimental necrotizing enterocolitis (NEC) was induced in newborn rats using a combination of gavage feeds and hypoxia. NHE isoforms were assessed in primary and cultured enterocytes by Western blot analysis and by confocal microscopy in the presence or absence of LPS. NHE activity was detected by single-cell fluorescent ratiometric imaging with the use of the pH-sensitive dye 2', 7'-bis-(2carboxyethyl) -5-(and-6)-carboxyfluorescein.


RESULTS
In both NEC and control rats, NHE1 was basolateral and NHE3 was apical. A similar distribution was observed in polarized IEC-6 cells. LPS caused a dose-dependent reduction in basolateral NHE1 activity in IEC-6 cells, but had no effect on apical NHE3 activity. This effect could not be accounted for by reduced expression or impaired plasma membrane localization of NHE isoforms. Strikingly, LPS-mediated NHE1 impairment caused marked cytoplasmic acidification under conditions of extracellular acidosis, whereas functional NHE1 maintained cytoplasmic pH homeostasis in control cells.


CONCLUSIONS
LPS selectively impairs basolateral NHE1 but not apical NHE3, leading to cytoplasmic acidification during extracellular acidosis. This effect could mediate impaired enterocyte function after LPS translocation and suggests a mechanism leading to barrier disruption in NEC.
Cetin
Selma
S
Division of Pediatric Surgery, Department of Surgery, Children's Hospital of Pittsburgh, University of Pittsburgh, and the Center of Biologic Imaging, PA 15213, USA.
Dunklebarger
Joshua
J
Li
Jun
J
Boyle
Patricia
P
Ergun
Orkan
O
Qureshi
Faisal
F
Ford
Henri
H
Upperman
Jeffrey
J
Watkins
Simon
S
Hackam
David J
DJ
D000138:Acidosis / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D020345:Enterocolitis, Necrotizing / Q000378:metabolism; D020895:Enterocytes / Q000187:drug effects* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008070:Lipopolysaccharides / Q000494:pharmacology*; D051381:Rats; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000187:drug effects*
D008070:Lipopolysaccharides; C000613387:Slc9a3 protein, rat; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; C069139:growth factor-activatable Na-H exchanger NHE-1