Signaling pathways of dicarboxylate transport in a proximal tubule cell line
The sodium-dicarboxylate co-transporter NaDC1/SLC13A2, located at the apical membrane of the proximal tubule, reabsorbs Krebs cycle intermediates from the glomerular filtrate and is thought to be responsible for the bulk of citrate transport in the nephron. Previous studies in our lab have shown that NaDC1 is not calcium-sensitive and that transport of citrate and succinate in opossum kidney (OK) proximal tubule cells is calcium-sensitive. When apical extracellular calcium is lowered apical citrate transport increases significantly in OK cells. In this study we demonstrate that stimulating the Calcium-Sensing Receptor (CaSR) by addition of spermine in normal and low extracellular calcium results in inhibition of succinate transport indicating that the CaSR plays a role in calcium-sensitive dicarboxylate transport regulation. Thapsigargin, commonly used to increase intracellular calcium, also inhibited succinate transport in normal and low extracellular calcium. This indicates that the CaSR signals through the guanine nucleotide binding protein Gq. In addition to increased intracellular calcium, Gq signaling also activates Protein Kinase C (PKC). The PKC activator, Phorbol 12-Myristate 13-Acetate (PMA), inhibited dicarboxylate transport in low extracellular calcium only indicating that PKC regulates calcium-sensitive transport specifically. Also investigated were the potential roles of the Gi and Gs pathways. Gi and Gs inhibit and activate adenylate cyclase respectively. Inhibiting Gi with Pertussis Toxin and treating cells with 8-Br-cAMP had no effect on transport indicating that neither Gi nor Gs regulate dicarboxylate transport. Inhibiting adenylate cyclase with MDL 12,330A decreased dicarboxylate transport in normal and low extracellular calcium. However, 8-Br-cAMP did not reverse these effects indicating that MDL 12,330A is inhibiting transport in some way other than Gi signaling. To determine that in OK cells if ligand biased signaling of the CaSR favors Gq at the expense of Gi and Gs, ELISA assays were used to measure intracellular cAMP concentration. We found that calcium and spermine do not change intracellular levels of cAMP. Thus, our studies on Gq, Gi and Gs signaling reveal that there is ligand biased signaling of the CaSR in favor of the Gq pathway and that dicarboxylate transport is regulated by the CaSR → Gq → PKC pathway.