Angiotensin II-induced apoptosis in rat cardiomyocyte culture: a possible role of AT(1) and AT(2) receptors

Objectives To investigate the mechanism of angiotensin II-induced apoptosis in cultured cardiomyocytes by determining which receptor subtype is involv ed, and what is the relationship between intracellular Ca2+ changes and apo ptosis. Design and methods Neonatal rat cardiomyocytes were pretreated with either the AT(1) antagonist irbesartan or the AT(2) antagonist PD123319 before exp osure to angiotensin Il. Apoptotis was evaluated using morphological techni que, staining nuclei by Feulgen and Hoechst methods followed by image analy sis and by in situ terminal deoxynucleotidyl transferase nick-end (TUNEL) l abelling. TUNEL-positive cardiocytes were distinguished from other cells by double staining with a-sarcomeric actin. Intracellular Ca2+ changes were a ssessed by indo-1 fluorescence microscopy, and the effect of Ca2+ on angiot ensin II-induced apoptosis was tested using the calcium channel blocker ver apamil. Results Exposure to angiotensin II (110 nmol/l) resulted in cell replicatio n and a three-fold increase in programmed cell death (P < 0.05). Pretreatme nt with either irbesartan (an AT(1) receptor antagonist, 100 nmol/l) or PD1 23319 (an AT(2) receptor antagonist, 1 mu mol/l) prevented the angiotensin II-induced apoptosis, indicating the presence of both AT(1) and AT(2) recep tors on cardiomyocytes. Exposure of myocytes to angiotensin II caused an im mediate and dose-dependent increase in the concentration of intracellular f ree Ca2+ that lasted 40-60 s. The effect was sustained in a Ca2+ free mediu m. Pretreatment of cells with irbesartan (100 nmol/l) and PD123319 (10 mu m ol/l) blocked Ca2+ elevation. Pretreatment with verapamil (10 lamol/1) prev ented angiotensin II-induced apoptosis. Conclusions Angiotensin II-induced apoptosis in rat cardiomyocytes is media ted through activation of both AT(1) and AT(2) receptors. The apoptotic mec hanism is not related to the immediate angiotensin II-induced Ca2+ rise fro m intracellular stores. However, it is accompanied by cardiomyocyte prolife ration and requires Ca2+ influx through L-type channel activity. (C) 2001 L ippincott Williams & Wilkins.