UBC Theses and Dissertations
Control of intracelluar Ca²⁺ in epithelial cells of the kidney thick ascending limb Dai, Long-jun
The cortical thick ascending limb of Henle’s loop (cTAL) plays a fundamental role in salt reabsorption and concentration-dilution processes within the nephron. This study was performed to characterize the role of intracellular free Ca²⁺ ([Ca²⁺]i) in hormone-mediated signal transduction and to describe the function of Na⁺/Ca²⁺ exchange in control of [Ca²⁺]i in isolated cTAL cells from porcine kidney. Parathyroid hormone, arginine vasopressin, and atrial natriuretic peptide transiently increased [Ca²⁺]i, in a dose-dependent manner. The increment in [Ca²⁺]i induced by these hormones was by intracellular release and entry through plasma membrane Ca²⁺ channels. These hormone-induced Ca²⁺ transients were modulated by cAMP, cGMP, and PKC activation. In order for intracellular Ca²⁺ to play a role in signal transduction mechanisms it is necessary to have regulated processes which maintain [Ca²⁺]i at submicromolar levels. We evaluated the functional role of Na⁺/Ca²⁺ exchange in cTAL cells. cTAL cells treated with ouabain had basal [Ca²⁺]i 86±2 nM. Removal of external Na⁺ or voltage depolarization with KC1 resulted in rapid and reversible maximal elevation of [Ca²⁺]i 1023 ±72 nM (n=28), which was dependent on the presence of external Ca²⁺ and elevated [Na⁺]i. The activity of Na⁺/ Ca²⁺ exchange was modulated by protein phosphorylation as calmodulin inhibition decreased and phosphatase inhibition increased the apparent exchange activity. The presence of a Na⁺/ Ca²⁺ exchanger was confirmed with northern hybridization techniques. A gene transcript which encodes a portion of the intracellular ioop of the renal Na⁺/ Ca²⁺ exchanger was amplified from cortical tissue and cTAL cells by polymerase chain reaction (PCR) using primers flanking the alternative splicing site. Southern hybridization and DNA sequencing demonstrated the isoform contained exons B and D characteristic of one isoform (NACA3) of the renal Na⁺/ Ca²⁺ exchanger. The results provide both functional and molecular evidence for a N2aICa exchanger in cTAL cells of the porcine kidney. It is likely that Na⁺/ Ca²⁺ exchange plays an important role in [Ca²⁺]i control and thus hormonal regulation of electrolyte reabsorption within the cTAL cells.