Intracellular ANG II induces cytosolic Ca2+ mobilization by stimulating intracellular AT1 receptors in proximal tubule cells.
Intracellular ANG II induces biological effects in nonrenal cells, but it is not known whether it plays a physiological role in renal proximal tubule cells (PTCs). PTCs express angiotensinogen, renin, and angiotensin-converting enzyme mRNAs, suggesting the presence of high levels of intracellular ANG II. We determined if microinjection of ANG II directly in single PTCs increases intracellular calcium concentration ([Ca2+]i) and, if so, elucidated the cellular mechanisms involved. Changes in [Ca2+]i responses were studied by fluorescence imaging using the Ca2+ indicator fluo 3. ANG II (1 nM) was microinjected directly in the cells, whereas cell-surface angiotensin type 1 (AT1) receptors were blocked by losartan (10 microM). When ANG II (1 nM) was added to the perfusate, there was a marked increase in [Ca2+]i that was blocked by extracellular losartan. With losartan in the perfusate, intracellular microinjection of ANG II elicited a robust increase in cytoplasmic [Ca2+]i that peaked at 30 s (basal: 2.2 +/- 0.3 vs. ANG II: 14.9 +/- 0.4 relative fluorescence units; P < 0.01). Chelation of extracellular Ca2+ with EGTA (2 mM) did not alter microinjected ANG II-induced [Ca2+]i responses (Ca2+ free + ANG II: 12.3 +/- 2.6 relative fluorescence units, not significant vs. ANG II); however, pretreatment with thapsigargin to deplete intracellular Ca2+ stores or with U-73122 to inhibit phospholipase C (1 microM each) markedly attenuated microinjected ANG II-induced [Ca2+]i responses. Combined microinjection of ANG II and losartan abolished [Ca2+]i responses, whereas a combination of ANG II and PD-123319 had no effect. These data demonstrate for the first time that direct microinjection of ANG II in single PTCs increases [Ca2+]i by stimulating intracellular AT1 receptors and releases Ca2+ from intracellular stores, suggesting that intracellular ANG II may play a physiological role in PTC function.
Zhuo
Jia L
JL
Division of Hypertension and Vascular Research, Henry Ford Hospital, 2799 West Grand Blvd., Detroit, MI 48202, USA. jzhuo1@hfhs.org
Li
Xiao C
XC
Garvin
Jeffrey L
JL
Navar
L Gabriel
LG
Carretero
Oscar A
OA
D000804:Angiotensin II / Q000032:analysis / Q000494:pharmacology*; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D007687:Kidney Tubules, Proximal / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D008845:Microinjections; D011817:Rabbits; D044140:Receptor, Angiotensin, Type 1 / Q000032:analysis / Q000187:drug effects / Q000502:physiology*; D044139:Receptor, Angiotensin, Type 2 / Q000187:drug effects / Q000502:physiology; D019284:Thapsigargin / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism
D044140:Receptor, Angiotensin, Type 1; D044139:Receptor, Angiotensin, Type 2; D000804:Angiotensin II; D019284:Thapsigargin; D010738:Type C Phospholipases; D002118:Calcium