Angiotensin II-induced apoptosis in rat cardiomyocyte culture: a possible role of AT1 and AT2 receptors.
OBJECTIVE
To investigate the mechanism of angiotensin II-induced apoptosis in cultured cardiomyocytes by determining which receptor subtype is involved, and what is the relationship between intracellular Ca2+ changes and apoptosis.


METHODS
Neonatal rat cardiomyocytes were pretreated with either the AT1 antagonist irbesartan or the AT2 antagonist PD123319 before exposure to angiotensin II. Apoptosis was evaluated using morphological technique, staining nuclei by Feulgen and Hoechst methods followed by image analysis and by in situ terminal deoxynucleotidyl transferase nick-end (TUNEL) labelling. TUNEL-positive cardiocytes were distinguished from other cells by double staining with alpha-sarcomeric actin. Intracellular Ca2+ changes were assessed by indo-1 fluorescence microscopy, and the effect of Ca2+ on angiotensin II-induced apoptosis was tested using the calcium channel blocker verapamil.


RESULTS
Exposure to angiotensin II (10 nmol/l) resulted in cell replication and a three-fold increase in programmed cell death (P < 0.05). Pretreatment with either irbesartan (an AT1receptor antagonist, 100 nmol/l) or PD123319 (an AT2 receptor antagonist, 1 micromol/l) prevented the angiotensin II-induced apoptosis, indicating the presence of both AT1 and AT2receptors on cardiomyocytes. Exposure of myocytes to angiotensin II caused an immediate and dose-dependent increase in the concentration of intracellular free Ca2+ that lasted 40-60 s. The effect was sustained in a Ca2+ free medium. Pretreatment of cells with irbesartan (100 nmol/l) and PD123319 (10 micromol/l) blocked Ca2+ elevation. Pretreatment with verapamil (10 micromol/l) prevented angiotensin II-induced apoptosis.


CONCLUSIONS
Angiotensin II-induced apoptosis in rat cardiomyocytes is mediated through activation of both AT1 and AT2 receptors. The apoptotic mechanism is not related to the immediate angiotensin II-induced Ca2+ rise from intracellular stores. However, it is accompanied by cardiomyocyte proliferation and requires Ca2+ influx through L-type channel activity.
Goldenberg
I
I
Internal Medicine D, Sheba Medical Center, Tel-Hashomer, Israel.
Grossman
E
E
Jacobson
K A
KA
Shneyvays
V
V
Shainberg
A
A
D000804:Angiotensin II / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D057911:Angiotensin Receptor Antagonists; D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D001713:Biphenyl Compounds / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002478:Cells, Cultured; D006321:Heart / Q000187:drug effects* / Q000502:physiology*; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism; D000077405:Irbesartan; D009206:Myocardium / Q000166:cytology; D009994:Osmolar Concentration; D051381:Rats; D017207:Rats, Sprague-Dawley; D044140:Receptor, Angiotensin, Type 1; D011945:Receptors, Angiotensin / Q000502:physiology*; D013777:Tetrazoles / Q000494:pharmacology; D014700:Verapamil / Q000494:pharmacology
D057911:Angiotensin Receptor Antagonists; D001713:Biphenyl Compounds; D002121:Calcium Channel Blockers; D044140:Receptor, Angiotensin, Type 1; D011945:Receptors, Angiotensin; D013777:Tetrazoles; D000804:Angiotensin II; D014700:Verapamil; D000077405:Irbesartan; D002118:Calcium