CHLORIDE‐INDEPENDENT, UNIDIRECTIONAL FLOW OF FLUID INTO THE SMALL INTESTINE: OPENING THE FLOODGATES
Authors: Buddington Randal K , Wong Thomas , Howard Scott C 
Published: November 2023

The volume of fluid in the gastrointestinal (GI) tract is the result of the carefully regulated dynamic balance between the counter processes of absorption and secretion. The ability to reversibly regulate fluid secretion is imperative for maintaining the normal gut fluid state. Diarrhea results from dysregulation of these processes and remains a leading cause of death worldwide. Although chloride secretion is generally considered as the mechanism for fluid secretion, it does not account for the large volumes of fluid and electrolytes that move into the intestinal lumen during diarrhea. We discovered using cultured freshly harvested mouse enterocytes in Ussing chamber studies a heretofore unreported unidirectional pathway for the movement of fluid into the small intestine. The fluid flow (FF) is directly related with mechanical force provided byaeration (none, minimal, intense, P<0.001), rapidly (<5 min) establishes apical to basolateral hydrostatic gradients that can exceed 2 cm that are sustained, is independent of ion gradients and channel inhibitors for Cl (P=0.4) and Na and K (P>0.50), does not require osmotic gradients, and is unidirectional based on movement of FITC‐dextran toward the apical chamber, but not from the apical to the basolateral chamber. The FF is related to extent of tight junction development based on the lack of FF by cultured fibroblasts (without tight junctions) and diminished FF by epithelia prepared using cells of colonic origin with extensive tight junction development (<20% for Caco‐2 and T84 cells compared with enterocytes). Exposure to zinc (100 microM) caused a rapid (<10 sec) inhibition of FF (>90%; P<0.001) that could be reversed by Zn chelation with citrate (~85% recovery; P<0.001). Sacs prepared from intact mouse small intestine were used to confirm cell data for FF based on loss of weight and leakage of FITC‐dextran. FF was increased by exposure to aeration and stirring (P=0.02), was higher for everted than uneverted sacs verifying FF is toward the lumen (P=0.02), and was inhibited ~40% by zinc (P=0.003).

Conclusions

The tight junction complex of enterocytes includes a “one‐way valve” for paracellular, unidirectional flow of fluid into the gut. This newly‐described paracellular pathway requires mechanical force that in vivo would be provided by contractions of smooth muscle, is sufficient to explain the massive fluid shifts that occur during diarrhea, and explains why the most effective anti‐diarrheal agents reduce smooth muscle contraction (and thus, mechanical force) and why agents that only inhibit chloride secretion have not proven effective. The reversible inhibition of FF by zinc, a safe and effective antidiarrheal agent, provides a promising target for therapeutics to treat diarrhea and constipation.

Hypotheses tested, experimental approaches, and results for fluid flow (FF) by cultured mouse enterocytes and sacs of intact small intestine.

Hypothesis
Experiment
Results

Experiments with cultured mouse enterocytes

FF requires aeration.
Record FF at no, low, and vigorous aeration
P<0.001 FF is related to aeration intensity

FF is unidirectional
Record movement of FF and FITC‐dextran when added to the apical vs the basolateral chamber
P<0.001 FF is paracellular and directed toward the lumen

FF is coupled with Cl flux
Record FF with Cl gradients and channel inhibitors
P>0.50 FF is independent of Cl flux

FF is coupled with Na or K flux
Record FF with Na and K gradients and inhibitors of channel and transporters
P>0.50 FF is independent of Na and K fluxes

FF responds to an osmotic gradient
Determine if an apical to basolateral osmotic gradient reduces FF
P>0.50 FF is independent of an osmotic gradient

Zinc inhibits FF
Measure FF before and after adding zinc sulfate
P<0.0001 zinc rapidly inhibits FF

Zinc inhibition of FF is reversible
Measure FF after adding citrate to chelate zinc
P<0.0001 citrate reverses Zn inhibition

Experiments with sacs prepared from mouse small intestine

Agitation increases FF
Record weight and FITC‐dextran leak with and without agitation by aeration and stirring
P<0.01 agitation increases weight loss and FITC‐dextran leakage

FF by intact tissue is unidirectional
Compare FF of evened and non‐everted sacs
P<0.01 FF greater for everted sacs

Zinc inhibits FF by intact tissue
Compare FF with and without zinc
P<0.001 zinc inhibits FF