Mechanisms of angiotensin II -: Mediated decreases in intraneuronal Ca2+ 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 [Ca2+] i, but complete blockade of Ang II effects was obtained with combined PKC and PKG inhibition. Modulation of inositol triphosphate (IP3)-inducible ER Ca2+ release by [Ca2+] i was investigated using furaptra, an ER-retaining dye. IP3-mediated ER Ca2+ release in beta-escin - permeabilized neurons was measured after clamping of [Ca2+] i from 50 nM to 800 nM. Maximal ER Ca2+ release was observed at approximate to 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 IP3R-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 Ca2+ release.