Several studies have demonstrated that angiotensin II (AII) directly influences electrolyte transport in renal proximal tubules. The effect is dose-dependent: Increased NaCl and NaHCO3 absorption at pM, decreased reabsorption at nM to mu M AII. the signalling mechanisms involved in these effects are not known. We have observed direct effects of AII on acutely isolated proximal tubule cells and primary cell cultures grown on porous membranes. We propose now to utilize the primary cell culture system of monolayers to evaluate whether luminal Na/H exchange and Cl conductance is regulated by AII and which intracellular messengers are involved. The effects of AII and putative intracellular messengers on transport will be assessed by electrophysiology, unidirectional and net fluxes of Na and Cl, and AII- dependent changes in intracellular pH and [Na] with fluorescent indicators, using video-enhanced light microscopy. The involvement of specific signalling pathways will be ascertained by determining the extent to which intracellular messengers (cAMP, eicosanoids, Ca) can explain changes in transport, either increases or decreases, depending on AII concentration. The levels of intracellular messengers will be manipulated independently of AII by pharmacological agents. To evaluate the effects of AII on transporter activity, Na and Cl transport will be measured in isolated brush border membrane vesicles isolated either from animals with different plasma AII levels (changes in salt balance, AII infusion, renal arterial stenosis) or from brush borders treated with putative regulatory enzymes (protein kinases, phosphatases). Na and Cl transport rates will be assessed under conditions of chemical equilibrium (isotope exchange) and energization by ion gradients. These experiments should clarify which electrolyte transporters are affected by different AII concentrations. In addition, intracellular signal that mediate the AII effects on transport will be identified. These studies will contribute importantly to our understanding of AII effects on renal functions and salt homeostasis and thereby provide insight into mechanisms of blood pressure regulation.
Showing the most recent 10 out of 110 publications
