Mechanisms of angiotensin II-mediated decreases in intraneuronal Ca ²⁺ in calcium-loaded stellate ganglion neurons

Our laboratory has reported previously that angiotensin II, type-1 (AT ₁) receptor stimulation in isolated stellate ganglion neurons decreases intraneuronal calcium concentration ([Ca²⁺]i) acutely if baseline [Ca²⁺]i is high and increases [Ca²⁺]i if baseline [Ca²⁺]i is low. Part of the angiotensin II (Ang II) effect in high Ca²⁺ neurons is mediated through stimulation of Na ⁺-Ca²⁺ exchange. Current experiments were conducted to identify additional steps in the signaling pathways. In Ca²⁺-loaded neurons, Ang II-induced decreases in [Ca²⁺]i were attenuated by phospholipase C inhibition (U73122) or nitric oxide (NO) synthase inhibition (L-NMMA) and were mimicked by the cGMP analogue 8-Br-cGMP. Protein kinase C (PKC) inhibition (bisindolylmaleimide I or Go6976) and protein kinase G (PKG) inhibition (KT5823) partially blocked Ang II-mediated decreases in [Ca ²⁺]i, but complete blockade of Ang II effects was obtained with combined PKC and PKG inhibition. Modulation of inositol triphosphate (IP ₃)-inducible ER Ca²⁺ release by [Ca²⁺]i was investigated using furaptra, an ER-retaining dye. IP₃-mediated ER Ca²⁺ release in β-escin-permeabilized neurons was measured after clamping of [Ca²⁺]i from 50 nM to 800 nM. Maximal ER Ca²⁺ release was observed at ≈200 nM [Ca²⁺]i, with noted blunting of release at higher [Ca²⁺]i. Steady-state mRNA transcript and protein levels revealed that the principal IP₃R isoform expressed was IP ₃R-II. These results suggest that Ca²⁺ loading in stellate ganglion neurons promotes Ang II-mediated decreases in [Ca²⁺]i via PKC and NO/cGMP/PKG pathways and inhibits IP₃R-II-mediated ER Ca ²⁺ release.