Astrocytes Reduce Store-Operated Ca2+ Entry in Microglia under the Conditions of an Inflammatory Stimulus and Muscarinic Receptor Blockade

Abstract

Inflammation and loss of cholinergic transmission are involved in neurodegenerative diseases, but possible interactions between them within neurons, astrocytes, and microglia have not yet been investigated. We aimed to compare store-operated Ca²⁺ entry (SOCE) in neurons, astrocytes, and microglia following cholinergic dysfunction in combination with (or without) an inflammatory stimulus and to investigate the effects of linalyl acetate (LA) on this process. We used the SH-SY5Y, U373, and BV2 cell lines related to neurons, astrocytes, and microglia, respectively. Scopolamine or lipopolysaccharide (LPS) was used to antagonize the muscarinic receptors or induce inflammatory responses, respectively. The concentration of intracellular Ca²⁺ was measured using Fura-2 AM. Treatment with scopolamine and LPS significantly increased SOCE in the neuron-like cells and microglia but not in the scopolamine-pretreated astrocytes. LA significantly reduced SOCE in the scopolamine-pretreated neuron-like cells and microglia exposed to LPS, which was partially inhibited by the Na⁺-K⁺ ATPase inhibitor ouabain and the Na⁺/Ca²⁺ exchanger (NCX) inhibitor Ni²⁺. Notably, SOCE was significantly reduced in the LPS plus scopolamine-pretreated cells mixed with astrocytes and microglia, with a two-fold increase in the applied number of astrocytes. LA may be useful in protecting neurons and microglia by reducing elevated SOCE that is induced by inflammatory responses and inhibiting the muscarinic receptors via Na⁺-K⁺ ATPase and the forward mode of NCX. Astrocytes may protect microglia by reducing increased SOCE under the conditions of inflammation and a muscarinic receptor blockade.

Keywords: astrocytes; linalyl acetate; lipopolysaccharide; microglia; scopolamine; store-operated Ca2+ entry.

Figure 1

Comparison of store-operated Ca²⁺ entry in SH-SY5Y, U373 and BV2 cells pretreated with scopolamine (SCOP) and treated with lipopolysaccharide (LPS), or treated with SCOP alone. Representative traces showing the effects of LPS in SCOP-pretreated (A) SH-SY5Y, (C) U373, and (E) BV2 cells. Summary data showing the peak [Ca²⁺]_i after adding 1.5 mM Ca²⁺ to the (B) SH-SY5Y, (D) U373, and (F) BV2 cells. Representative traces showing effects of SCOP alone to the (G) SH-SY5Y, (I) U373, and (K) BV2 cells. Summary data showing the peak [Ca²⁺]_i after adding 1.5 mM Ca²⁺ to the (H) SH-SY5Y, (J) U373 and (L) BV2 cells. Data are reported as means ± SEM (n = 6–10; ^## p < 0.01 vs. the control (CON) group.

Figure 2

Effects of linalyl acetate (LA) on store-operated Ca²⁺ entry (SOCE) in SH-SY5Y and BV2 cells pretreated with scopolamine (SCOP) and treated with lipopolysaccharide (LPS) and the hypothesized mechanism underlying the LA-induced reduction in SOCE in LPS-exposed, SCOP-pretreated SH-SY5Y and BV2 cells. Representative traces showing the effects of LA on SOCE in scopolamine-pretreated and LPS-exposed (A) SH-SY5Y and (E) BV2 cells. Summary data showing the peak [Ca²⁺]_i observed after the addition of 1.5 mM Ca²⁺ in (B) SH-SY5Y and (F) BV2 cells. Representative traces showing the effect of ouabain, Ni²⁺, or nifedipine on the LA-induced decreases in SOCE of LPS-exposed, SCOP-pretreated (C) SH-SY5Y and (G) BV2 cells. Summary data showing the peak [Ca²⁺]_i after adding 1.5 mM Ca²⁺ in (D) SH-SY5Y and (H) BV2 cells. Data are reported as means ± SEM (n = 5–9; ^### p < 0.001 vs. the control (CON) group; ** p < 0.01, *** p < 0.001 vs. the SCOP+LPS group; ⁺ p < 0.05, ⁺⁺ p < 0.01, ⁺⁺⁺ p < 0.001 vs. the SCOP + LPS + LA group).

Figure 3

Effects of doubling the number of U373 cells on store-operated Ca²⁺ entry (SOCE) in scopolamine (SCOP)-pretreated SH-SY5Y + U373 + BV2 mixed cells exposed to lipopolysaccharide (LPS), and the hypothesized mechanism by which the doubling in number of U373 cells reduced SOCE in LPS-exposed, SCOP-pretreated SH-SY5Y + U373 + BV2 mixed cells. Representative traces showing the effects of doubling the number of U373 cells on SOCE in the (A) control (CON) and (B) SCOP+LPS and (C) SCOP groups. (D) Summary data showing the peak [Ca²⁺]_i observed after the addition of 1.5 mM Ca²⁺. (E) Representative traces showing the effect of ouabain, Ni²⁺, or nifedipine on the doubled number of U373 cells-induced decreases in SOCE in LPS-exposed, SCOP-pretreated SH-SY5Y + U373 + BV2 mixed cells. (F) Summary data showing the peak [Ca²⁺]_i observed after the addition of 1.5 mM Ca²⁺. Data are reported as means ± SEM (n = 5–9; ^# p < 0.05, ^### p < 0.001 vs. the respective CON group; ** p < 0.01, *** p < 0.001 vs. the respective vehicle group; ⁺ p < 0.05 vs. the SCOP+LPS group in doubled U373 cells).

Figure 4

Effects of doubling the number of U373 cells on store-operated Ca²⁺ entry (SOCE) in scopolamine (SCOP)-pretreated U373 + BV2 mixed cells exposed to LPS. Representative traces of the (A) control (CON), (B) SCOP+LPS, and (C) SCOP groups. (D) Summary data showing the peak [Ca²⁺]_i observed after the addition of 1.5 mM Ca²⁺. Data are reported as means ± SEM (n = 5–7; ^## p < 0.01 vs. the respective CON group; * p < 0.05 vs. the respective vehicle group).

Figure 5

Proposed mechanisms of action of linalyl acetate (LA) on store-operated Ca²⁺ entry (SOCE) in SH-SY5Y and BV2 cells exposed to scopolamine and lipopolysaccharide (LPS). Red arrows reflect the impact of scopolamine and LPS, and blue arrows reflect the effects of LA. The amount of Ca²⁺ influx or efflux is indicated by the thicknesses of the arrows. IP₃R, inositol 1,4,5-trisphosphate receptor; NCX, Na⁺/Ca²⁺ exchanger; RyR, ryanodine receptor; SERCA, sarcoplasmic reticulum Ca²⁺ ATPase; SOC, store-operated Ca²⁺ channel.