Angiotensin II Regulation of Intracellular Calcium in Astroglia Cultured from Rat Hypothalamus and Brainstem

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Abstract
This study examines the angiotensin II (Ang II) regulation of intracellular free calcium concentration ([Ca2+]i) in astroglia cultured from the hypothalamus and brainstem of the adult rat. Bath perfusion or rapid puffer application of angiotensin II (Ang II) (1–100 nM) increased [Ca2+]i in both polygonal and stellate astroglia when measured using fura-2 imaging fluorescence microscopy. Ang II increased [Ca2+]i in 96.1 and 95.6% of the polygonal and stellate glial cells, respectively. In normal Tyrode's solution (containing 2 mM CaCl2), the Ang II-stimulated increase in [Ca2+]i characteristically showed a biphasic response, i.e., an initial rapid transient peak followed by a sustained, steady-state plateau of free Ca2+. In both cell types, the selective Ang II type 1 receptor subtype (AT1) antagonist losartan (1 µM) inhibited the Ang II-stimulated increase in [Ca2+]i. The selective AT2 antagonist PD 123319 (1 µM) did not inhibit the Ang II-stimulated increase in [Ca2+]i in either cell type. To define the sources of Ca2+ that participate in the Ang II-stimulated increase in [Ca2+]i in astroglia, experiments were performed in a nominally Ca2+-free Tyrode's solution. In either cell type, this resulted in only an initial transient increase of Ca2+ and no sustained plateau of Ca2+ when challenged with Ang II. Thapsigargin (5 µM), cyclopiazonic acid (10 µM), and ryanodine (10 µM), but not caffeine (1–10 mM), inhibited the initial rise in [Ca2+]i. The plateau increase of [Ca2+]i caused by Ang II (100 nM) was reversibly inhibited by both cadmium (100 µM) and nifedipine (10 µM); in contrast, gadolinium (100 µM) had no effect on the plateau increase of [Ca2+]i. These results indicate that Ang II, in physiological concentrations, can activate AT1 receptors to stimulate both Ca2+ release from intracellular stores and Ca2+ influx from the extracellular space to increase [Ca2+]i of polygonal and stellate astroglia.

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