Individual Amino Acids Differentially Regulate Paracellular Calcium Flux across an Intestinal Cell Model

Abstract

Acute high protein intake results in increased intestinalcalcium absorption in humans and animals. Calcium is absorbed through both passive paracellular and facilitated trans cellular pathways. In rats, the increase in intestinal calcium absorption observed during a high protein dietis mediated at least in part through increased apical intestinal calcium uptake suggesting involvement of the transcellular pathway. However, we previously found that the expression of a paracellular calcium‐permissive tight junction protein, Claudin‐2, is higher in the duodenum of rats consuming a high compared to lowprotein diet. Whether dietary protein affects paracellular calcium flux has not been evaluated. Furthermore, the bioactive amino acids that might underlie this effect have not been identified. The purpose of this study was to evaluate the direct effects of mixed and individual amino acids on paracellular calcium flux across a small intestinal absorptive cell model.MethodsCaco‐2 Bbe cells were used as a model of small intestinal absorptive epithelial cells. Confluent monolayers were grown on snap well inserts and mounted in Ussing chambers for flux studies under a mucosal to serosal calcium gradient to simulatepost‐prandial conditions. Unidirectionalcalcium flux over 90 min was calculated using 44Ca as a tracer with serosal samples collected every 30 min. Treatments were applied acutely to bothmucosal and serosal chambers and included vehicle (no treatment control), 80mMraffinose as a positive control, mixed amino acids, and 2, 5 and 10mMleucine, phenylalanine and lysine. RNA expression of calcium permissive tight junction proteins was measured using qPCR.ResultsLinear regression analysis modeling Ca flux (nmol/cm2/hr) by treatment using vehicle control as the reference group elicited significant increases with 80mM raffinose (β= 66.7, t= 3.2, p< 0.05) and 5mM leucine (β= 67.6, t= 1.9, p≤0.05), whereas 10mM phenylalanine resulted in a significant decrease in Ca flux (β= −89.3, t=−1.9, p< 0.05). No significant changes were observed with mixed amino acidor lysine treatment. The effects of amino acids on Ca flux were not associated with changes in mRNA expression of the Ca permissive tight junction proteins claudins‐2 and 12.ConclusionsThese data demonstrate that amino acids have differential effects on paracellular Ca flux in a model of intestinalabsorptive epithelia. This suggests that specific amino acid constituents of dietary protein may be partially responsible for protein‐mediated increases inintestinal Ca absorption. Gene expression of claudins‐2 and 12 did not explain aminoacid induced changes in Ca flux. Future research will investigate posttranscriptional effects of amino acids on tight junction proteins in anintestinal epithelial model.Support or Funding InformationResearch supported by: USAMRMC. The views expressed in this abstract are those of the authors and do not reflect the official policy of the Department of Army, Department of Defense, or the U.S. Government.