Calcium and protein kinase C modulate ion transport in numerous cells but their roles in the renal proximal tubule have not been clearly defined. Therefore, this project proposes an in-depth study of calcium regulation of sodium and potassium transport in the mammalian proximal tubule. Initial studies will directly test the theory of negative feedback control of sodium transport by cytosolic calcium (39,40). Subsequently, the ability of cytosolic calcium and protein kinase C activation to alter luminal sodium entry and basolateral sodium and potassium transport will be investigated. Finally, time permitting, the roles of calcium and protein kinase C in the regulation of sodium and potassium transport following a physiological perturbation (i.e., glucose addition) will be determined. Techniques such as nuclear magnetic resonance (NMR) spectroscopy to monitor intracellular sodium and pH, extracellular ion-sensitive electrodes to monitor net fluxes of potassium and volumes changes, and fluorescence spectroscopy (fura-2) to measure cytosolic calcium will be used to distinguish the effects of selected perturbations of cellular calcium metabolism on transport in suspensions of rabbit proximal tubules. The following specific aims will be addressed: 1) Is cytosolic calcium inversely related to ouabain-sensitive respiration, net potassium uptake, and intracellular sodium? 2) Do phorbol esters or changes in cytosolic calcium alter luminal Na+/H+ exchange, Na+K+-ATPase turnover, and passive potassium permeability? NMP observation of intracellular sodium and pH combined with potassium flux measurements will address these issues. 3) How does luminal Na+-glucose cotransport alter the intracellular levels and transport of sodium, potassium, and calcium? Investigation of the regulation of ion transport in the intact mammalian proximal tubule has been largely confined to measurements of transepithelial transport. Recent advances in cellular physiology, however, have emphasized the need to observe intracellular events in order to evaluate basic cellular function. Therefore, this investigation will apply powerful new techniques to observe intracellular ions and thereby provide a fundamental understanding of the regulatory roles of calcium and protein kinase C in an ion transporting epithelium. Given the ubiquitous nature of calcium as a cellular regulator, the results of this investigation will have direct implications for numerous aspects of cellular physiology.
