Exposure of luminal membranes of LLC-PK₁cells to ANG II induces dimerization of AT₁/AT₂receptors to activate SERCA and to promote Ca²⁺mobilization

Abstract

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...

L-Tyr-Induced Phosphorylation of Tyrosine Hydroxylase at Ser⁴⁰: An Alternative Route for Dopamine Synthesis and Modulation of Na⁺/K⁺-ATPase in Kidney Cells

“…We avoid using cell cultures after the 30 th passage for the different experimental conditions; these cells had adhered to the flask by the basolateral side, thus keeping their polarity, an important characteristic of this cell line [22, 23, 24, 25]. To ascertain the effects of L-tyr and L-dopa on the ATPase activity, cells were incubated with them in Hank’s solution (137 mM NaCl, 5 mM KCl, 0.8 mM MgSO 4 , 1 mM MgCl 2 , 0.33 mM Na 2 HPO 4 , 0.44 mM KH 2 PO 4 , 0.2 mM Tris-HCl and 20 mM Hepes 0.25 mM CaCl 2 , pH 7.4), alone, or in the presence of the LAT2 inhibitor (BCH) and the D 1 R antagonist (Sch 23390).…”

Renin-angiotensin system in the kidney: What is new?

“…In vitro studies using isolated basolateral membrane fractions from pig kidney have demonstrated that Ang II stimulates the renal proximal tubule Na + -ATPase activity via PI-PLCb/PKC pathway [99,100] . It is widely known that intracellular Ca 2+ mobilization in proximal tubule cells leads to the activation of many Ca 2+ -dependent intracellular signaling pathways, including those associated with Na + reabsorption [101] . Ang Ⅱ microperfusion techniques in rabbit superficial segment of proximal tubules in vitro regulated Na + reabsorption via PKC and intracellular Ca 2+ [102] ; low concentrations of Ang Ⅱ inhibited membrane Ca 2+ -ATPase via AT1/AT2 receptors heterodimers and PKC in isolated fractions of basolateral membranes of proximal tubule, increasing cytosolic Ca 2+ concentration in proximal tubule cells [37,103] .…”

“…Ang Ⅱ microperfusion techniques in rabbit superficial segment of proximal tubules in vitro regulated Na + reabsorption via PKC and intracellular Ca 2+ [102] ; low concentrations of Ang Ⅱ inhibited membrane Ca 2+ -ATPase via AT1/AT2 receptors heterodimers and PKC in isolated fractions of basolateral membranes of proximal tubule, increasing cytosolic Ca 2+ concentration in proximal tubule cells [37,103] . Luminal Ang Ⅱ stimulates AT1/AT2 receptors heterodimerization that increases sarco/endoplasmic reticulum Ca 2+ -ATPase activity and promotes Ca 2+ mobilization in proximal tubule cells [101] . The intracrine/intracellular system is new paradigm.…”

“…An alternative way for the intracellular source of Ang Ⅱ is the internalization of extracellular Ang Ⅱ after binding to the AT1 surface receptor [82,83] . Not all internalized Ang Ⅱ-AT1 complex is degraded in lysosomes, thereby increasing its concentration within the cell, and the AT1 receptor may be relocated to other organelles, including the nucleus [101,[104][105][106][107][108] . Indeed, subcellular localization of 125 I-labeled Ang Ⅱ in the pig kidney indicates that Ang Ⅱ generation is predominantly extracellular, followed by AT1 receptor-mediated endocytosis leading to higher intracellular Ang Ⅱ levels [109] .…”

Synergy of antihypertensives in elderly patients with CKD

“…6 Improved calcium handling may, in turn, lead to improvements in diastolic and systolic function of the heart, improve vascular relaxation and may even improve proximal tubule function. 7,8 …”