Cholinergic signaling inhibits oxalate transport by human intestinal T84 cells

Abstract

Urolithiasis remains a very common disease in Western countries. Seventy to eighty percent of kidney stones are composed of calcium oxalate, and minor changes in urinary oxalate affect stone risk. Intestinal oxalate secretion mediated by anion exchanger SLC26A6 plays a major constitutive role in limiting net absorption of ingested oxalate, thereby preventing hyperoxaluria and calcium oxalate urolithiasis. Using the relatively selective PKC-δ inhibitor rottlerin, we had previously found that PKC-δ activation inhibits Slc26a6 activity in mouse duodenal tissue. To identify a model system to study physiologic agonists upstream of PKC-δ, we characterized the human intestinal cell line T84. Knockdown studies demonstrated that endogenous SLC26A6 mediates most of the oxalate transport by T84 cells. Cholinergic stimulation with carbachol modulates intestinal ion transport through signaling pathways including PKC activation. We therefore examined whether carbachol affects oxalate transport in T84 cells. We found that carbachol significantly inhibited oxalate transport by T84 cells, an effect blocked by rottlerin. Carbachol also led to significant translocation of PKC-δ from the cytosol to the membrane of T84 cells. Using pharmacological inhibitors, we observed that carbachol inhibits oxalate transport through the M(3) muscarinic receptor and phospholipase C. Utilizing the Src inhibitor PP2 and phosphorylation studies, we found that the observed regulation downstream of PKC-δ is partially mediated by c-Src. Biotinylation studies revealed that carbachol inhibits oxalate transport by reducing SLC26A6 surface expression. We conclude that carbachol negatively regulates oxalate transport by reducing SLC26A6 surface expression in T84 cells through signaling pathways including the M(3) muscarinic receptor, phospholipase C, PKC-δ, and c-Src.

Extracellular nucleotides inhibit oxalate transport by human intestinal Caco-2-BBe cells through PKC-δ activation

“…Purinergic signaling modulates many intestinal processes through pathways including PKC activation (12), which we previously demonstrated to inhibit intestinal oxalate transport (38,39). We therefore examined whether purinergic stimulation with the extracellular nucleotide ATP regulates oxalate transport by C2 cells.…”

“…T84 cells were obtained from the American Type Culture Collection (Manassas, VA) and grown in DMEM-Ham's F-12 culture medium supplemented with 5-10% FBS, 50 U/ml penicillin, and 50 mg/ml streptomycin at 37°C in 5% CO 2-95% air. The oxalate flux and other studies described below were performed using confluent cells grown for 5-13 days postplating on 24-well plastic supports and/or on 0.4-m collagen-coated polystyrene Transwell membrane filters (Corning, Corning, NY) in 12-and/or 24-mm inserts after overnight serum starvation (11,13,38,76). C2 cells were similarly grown on 0.4-m collagen-coated 12-mm Snapwell inserts (Corning) for the Ussing chamber studies.…”

“…The transepithelial resistance (TER) was measured using an epithelial voltohmmeter (EVOM2, World Precision Instruments) to monitor the maturation of the monolayers grown on Transwell permeable supports. We also assessed villin protein expression (38), a marker of epithelial maturation (4,30), to monitor differentiation of the monolayers. This was done by harvesting total protein lysates from C2 cells on days 1, 2, 3, 4, 5, 6, 8, 10, 12, and 14 postplating for determination of villin protein expression using immunoblotting.…”

“…Cells were treated with vehicle (control) or 100 M ATP for 30 min in the culture medium and then washed once in ice-cold PBS. C2 cells were subjected to two 30-min incubations in the biotinylation reagent Sulfo-NHS-SS-Biotin (Thermo Scientific; 2 mg/ml) in PBS (38,39) to biotinylate apical membrane proteins (with all steps performed on ice and/or in a cold room) and washed twice in ice-cold PBS. The cells were then incubated in PBS containing 100 mM glycine for 15 min to quench excess biotin and washed twice with PBS.…”

Pediatric primary urolithiasis: Symptoms, medical management and prevention strategies

“…The development of new techniques allowed the conceivement future of alternative treatments for cystinuria. Inhibition of the cystine transporter [120] and of the cystine crystal growth (Lcystine dimethyl ester_L CDME) are promising measures to prevent cystine Urolithiasis [121,122] . They appear to be effective even at low concentrations, improving the safety profile of this sort of treatment.…”