Tu1401 Differential Intracellular Calcium Inhibition of NHE3 Activity

Abstract

BACKGROUND: Elevation of intracellular calcium ([Ca]i) in intestinal epithelial cells occurs as part of normal digestion resulting in a transient decrease of NaCl absorption and increase in anion secretion. Under pathophysiological conditions, particularly rotavirus (RV) infection, elevation of [Ca]i is responsible for decreased Na+ absorption, increased Clsecretion and diarrhea. We have shown that elevated [Ca]i by either carbachol (CCH) or RV decreases NHE3 activity by stimulating rapid endocytosis; however, the differential signaling mechanisms responsible for the severe water loss due to RV infection are unclear. PURPOSE: Since NHE3 contributes to RV diarrhea, the current goal was to compare how elevated [Ca]i signaling differs under physiological (CCH) and pathophysiological (RV) conditions and determine their effects on NHE3. METHODS: Caco-2/BBe cells were grown on filters and transiently infected with a 3HA-NHE3 adenovirus construct and studied 48 h later. NHE3 activity was measured by fluorimetry using pH-sensitive dye, BCECF, in cells treated with vehicle, BAPTA-AM, latrunculin, methyl-β-cylcodextrin (MβCD), or inhibitors to PLD1 (EVJ), PLD2 (JWJ), Cdc42 (Pirl1), or acid sphingomyelinase (ASM; imipramine), in the presence or absence of CCHor simian rotavirus, RRV.NHE3 trafficking and degradation were determined by surface biotinylation. RESULTS: Both CCH and RV inhibited NHE3 activity by ~60% in a Ca2+ dependent manner. These effects were correlated with >50% decrease in NHE3 surface expression due to increased NHE3 endocytosis. NHE3 exocytosis was not different after CCH compared to control; however, NHE3 exocytosis was greatly reduced after RV infection. In addition, RV, but not CCH, treatment decreased NHE3 halflife. Pretreatment with either Pirl1, MβCD, both PLD inhibitors, imipramine, or latrunculin prevented CCH-mediated inhibition of NHE3 activity. In contrast, only MβCD and JWJ prevented RV inhibition of NHE3 while latrunculin had additive effects on NHE3 (>90% inhibition) after RV infection.CONCLUSIONS: (1) Physiologic and pathophysiologic [Ca]i elevation has differential effects on NHE3 regulation. (2) While both CCH and RV inhibit apical NHE3 activity through changes in trafficking due to elevated [Ca]i, RV also inhibits NHE3 activity by decreasing protein amount possibly due to a block in apical recycling since RV prevented NHE3 exocytosis. (3) These effects involve lipid rafts since cholesterol depletion and ceramide production by ASM prevent NHE3 inhibition; however, actin remodeling involving Cdc42 was not required for RV inhibition of NHE3 suggesting that RV infection stimulates a different endocytosis pathway for NHE3 compared to CCH resulting in increased NHE3 protein degradation. (4) Rotavirus infection is a new model to dissect mechanisms that regulate NHE3 activity in intestinal epithelial cells.