Using the PLC inhibitor U73122, PMA, and calphostin C, it was possible to demonstrate the involvement of a PLC¡DAG(PMA)¡PKC pathway in the stimulation of SERCA by ANG II with no effect on PMCA. We conclude that ANG II triggers SERCA activation via the luminal membrane, increasing the Ca 2ϩ stock in the reticulum to ensure a more efficient subsequent mobilization of Ca 2ϩ . This first report on the regulation of SERCA activity by ANG II shows a new mechanism for Ca 2ϩ homeostasis in renal cells and also for regulation of Ca 2ϩ -modulated fluid reabsorption in proximal tubules.luminal effect of ANG II; Ca 2ϩ sparks; proximal tubule Ca 2ϩ homeostasis; fluid reabsorption THE KIDNEY IS an essential organ in homeostasis and body fluid regulation. Its functions are modulated by different hormones and autacoids that act on hydroelectrolytic balance, extracellular volume, and blood pressure, such as angiotensin II (ANG II) (24). ANG II plays a crucial role in renal Ca 2ϩ handling, one of the main cell messengers (4, 5, 13, 51), which is involved in the fine tuning of fluid reabsorption in different segments of the nephron (17). Approximately 60% of the plasma Ca 2ϩ is filtered by the kidneys, 99% of which is reabsorbed (70% of it in the proximal tubules) (19). ANG II regulates Ca 2ϩ reabsorption in both luminal and basolateral aspects of tubule membranes (4, 5, 13). In proximal tubule cells, intracellular Ca 2ϩ mobilization activates Ca 2ϩ -dependent intracellular signaling pathways, including those associated with ANG II-modulated Na ϩ and water reabsorption mechanisms (8,16,17,39,42).Active and passive transport mechanisms participate in Ca 2ϩ reabsorption in the proximal tubules (19,48). Concerning primary active transporters, there are two main Ca 2ϩ -ATPase subfamilies in proximal tubule cells, the sarco(endo)plasmic reticulum Ca 2ϩ -ATPase (SERCA) and the plasma membrane Ca 2ϩ -ATPase (PMCA) (38). These pumps control intracellular Ca 2ϩ concentration by regulating intracellular Ca 2ϩ stocks, fine-tuning cytosolic Ca 2ϩ activity in many cell types (12). Therefore, renal SERCA and PMCA are excellent targets for hormones, including ANG II and angiotensin-derived peptides (6). We found that picomolar ANG II inhibits PMCA activity via a PLC/protein kinase C (PKC) pathway triggered by the peptide binding to AT 1 R/AT 2 R heterodimers (4, 5). Micromolar ANG II is metabolized by peptidases associated with the basolateral membrane, generating Ang-(3-4) that reactivates PMCA (5, 6).Classically, ANG II was described to be formed only systemically. Different studies in the last two decades found that ANG II is formed in different tissues such as brain, heart, and adipose tissue (7,18,34) and, especially, in kidney, where local renin-angiotensin system was first described (10,20,25,26,28,50). Because of this local synthesis, ANG II levels are much higher in the kidney than in plasma (37, 43), being found in proximal tubule fluid in the nanomolar range (9,23,36,43). This tubular fluid ANG II is not only derived from the filtrate...

The physiological roles of ANG-(3-4) (Val-Tyr), a potent ANG II-derived peptide, remain largely unknown. The present study 1)investigates whether ANG-(3-4) modulates ouabain-resistant Na(+)-ATPase resident in proximal tubule cells and 2) verifies whether its possible action on pumping activity, considered the fine tuner of Na(+) reabsorption in this nephron segment, depends on blood pressure. ANG-(3-4) inhibited Na(+)-ATPase activity in membranes of spontaneously hypertensive rats (SHR) at nanomolar concentrations, with no effect in Wistar-Kyoto (WKY) rats or on Na(+)-K(+)-ATPase. PD123319 (10(-7) M) and PKA(5-24) (10(-6) M), an AT2 receptor (AT2R) antagonist and a specific PKA inhibitor, respectively, abrogated this inhibition, indicating that AT2R and PKA are central in this pathway. Despite the lack of effect of ANG-(3-4) when assayed alone in WKY rats, the peptide (10(-8) M) completely blocked stimulation of Na(+)-ATPase induced by 10(-10) M ANG II in normotensive rats through a mechanism that also involves AT2R and PKA. Tubular membranes from WKY rats had higher levels of AT2R/AT1R heterodimers, which remain associated in 10(-10) M ANG II and dissociate to a very low dimerization state upon addition of 10(-8) M ANG-(3-4). This lower level of heterodimers was that found in SHR, and heterodimers did not dissociate when the same concentration of ANG-(3-4) was present. Oral administration of ANG-(3-4) (50 mg/kg body mass) increased urinary Na(+) concentration and urinary Na(+) excretion with a simultaneous decrease in systolic arterial pressure in SHR, but not in WKY rats. Thus the influence of ANG-(3-4) on Na(+) transport and its hypotensive action depend on receptor association and on blood pressure.