Exposure of luminal membranes of LLC-PK1 cells to ANG II induces dimerization of AT1/AT2 receptors to activate SERCA and to promote Ca2+ mobilization.
ANG II is secreted into the lumens of proximal tubules where it is also synthesized, thus increasing the local concentration of the peptide to levels of potential physiological relevance. In the present work, we studied the effect of ANG II via the luminal membranes of LLC-PK(1) cells on Ca(2+)-ATPase of the sarco(endo)plasmic reticulum (SERCA) and plasma membrane (PMCA). ANG II (at concentrations found in the lumen) stimulated rapid (30 s) and persistent (30 min) SERCA activity by more than 100% and increased Ca(2+) mobilization. Pretreatment with ANG II for 30 min enhanced the ANG II-induced Ca(2+) spark, demonstrating a positively self-sustained stimulus of Ca(2+) mobilization by ANG II. ANG II in the medium facing the luminal side of the cells decreased with time with no formation of metabolites, indicating peptide internalization. ANG II increased heterodimerization of AT(1) and AT(2) receptors by 140%, and either losartan or PD123319 completely blocked the stimulation of SERCA by ANG II. 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(2+) stock in the reticulum to ensure a more efficient subsequent mobilization of Ca(2+). This first report on the regulation of SERCA activity by ANG II shows a new mechanism for Ca(2+) homeostasis in renal cells and also for regulation of Ca(2+)-modulated fluid reabsorption in proximal tubules.
Instituto de Biofísica Carlos Chagas Filho, Laboratório de Físico-Química Biológica Aída Hassón-Voloch, Universidade Federal do Rio de Janeiro, Avenida Carlos Chagas Filho 373, Rio de Janeiro, Brazil.
Fernanda M
FM
Ferrão
Lucienne S
LS
Lara
Flavia
F
Axelband
Juliana
J
Dias
Adriana K
AK
Carmona
Rosana I
RI
Reis
Claudio M
CM
Costa-Neto
Adalberto
A
Vieyra
Jennifer
J
Lowe
D000804:Angiotensin II / Q000378:metabolism*; D000085962:Angiotensin-Converting Enzyme 2; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D007687:Kidney Tubules, Proximal / Q000378:metabolism*; D015260:Neprilysin / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism; D007703:Peptidyl-Dipeptidase A / Q000378:metabolism; D055503:Protein Multimerization; D044140:Receptor, Angiotensin, Type 1 / Q000378:metabolism*; D044139:Receptor, Angiotensin, Type 2 / Q000378:metabolism*; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism*; D015398:Signal Transduction; D013552:Swine
D044140:Receptor, Angiotensin, Type 1; D044139:Receptor, Angiotensin, Type 2; D000804:Angiotensin II; D010447:Peptide Hydrolases; D007703:Peptidyl-Dipeptidase A; D000085962:Angiotensin-Converting Enzyme 2; D015260:Neprilysin; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D002118:Calcium