Loose excitation-secretion coupling in astrocytes

Abstract

Astrocytes play an important housekeeping role in the central nervous system. Additionally, as secretory cells, they actively participate in cell-to-cell communication, which can be mediated by membrane-bound vesicles. The gliosignaling molecules stored in these vesicles are discharged into the extracellular space after the vesicle membrane fuses with the plasma membrane. This process is termed exocytosis, regulated by SNARE proteins, and triggered by elevations in cytosolic calcium levels, which are necessary and sufficient for exocytosis in astrocytes. For astrocytic exocytosis, calcium is sourced from the intracellular endoplasmic reticulum store, although its entry from the extracellular space contributes to cytosolic calcium dynamics in astrocytes. Here, we discuss calcium management in astrocytic exocytosis and the properties of the membrane-bound vesicles that store gliosignaling molecules, including the vesicle fusion machinery and kinetics of vesicle content discharge. In astrocytes, the delay between the increase in cytosolic calcium activity and the discharge of secretions from the vesicular lumen is orders of magnitude longer than that in neurons. This relatively loose excitation-secretion coupling is likely tailored to the participation of astrocytes in modulating neural network processing.

Keywords: GPCR; SNAREs; astrocyte secretion; calcium homeostasis; exocytosis; vesicular release.

Figure 1. Sources of Ca²⁺ for regulated vesicle-based secretion from astrocytes

The accumulation of Ca²⁺ in the cytosol may occur (1) following the entry of Ca²⁺ from the extracellular space (ECS) through L-type voltage-gated channels (VGCC), store-operated Ca²⁺ entry (SOCE) via transient receptor potential canonical type 1-containing channels, and the plasma membrane Na⁺/Ca²⁺ exchanger (NCX), and (2) via G protein-coupled receptor (GPCR) activation, which can generate second messengers cAMP, inositol 1,4,5 triphosphate (IP₃), and diacylglycerol (DAG). The GPCR activation in astrocytes retrieves Ca²⁺ from the ER internal stores that possess IP₃ receptors (IP₃R) as well as from ryanodine (Ry)-sensitive channels acting as conduits for Ca²⁺ delivery to the cytosol. The ER store is (re)filled by Ca²⁺-ATPase (i.e., SERCA pumps), which can be blocked by thapsigargin (Thaps). Cytosolic Ca²⁺ levels are modulated by mitochondria. These organelles take up Ca²⁺ via the Ca²⁺ uniporter, which is blocked by ruthenium 360 (Ru360), during the cytosolic Ca²⁺ increase. As cytosolic Ca²⁺ decreases due to the extrusion mechanisms, Ca²⁺ is slowly released by mitochondria into the cytosol via the mitochondrial Na⁺/Ca²⁺ exchanger as well as by the transient opening of the mitochondrial permeability transition pore. This transient opening is indirectly blocked by cyclosporin A (CsA), which binds cyclophilin D (not shown). The increase in cytosolic Ca²⁺ levels is sufficient and necessary to cause the fusion of secretory vesicles (which themselves can act as IP₃-sensitive stores for Ca²⁺) with the plasma membrane, mediating the exit of gliosignaling molecules (such as amino acids, peptides, and ATP) from the vesicle lumen into the ECS. The cAMP-mediated modulation of Ca²⁺ homeostasis may occur at the level of Ca²⁺ entry or extrusion from the cytosol. Moreover, cAMP-mediated mechanisms may directly affect the fusion pore and the extrusion of gliosignals from the vesicle lumen. Drawing is not to scale.

Figure 2. Delay between the increase in cytosolic Ca²⁺ and the increase in membrane capacitance reported for vesicle-based secretory activity in astrocytes

(A) Typical responses of membrane capacitance (C_m) in three individual astrocytes elicited by flash (UV) photolysis-induced (upward-pointing arrow) elevations in the intracellular calcium concentration ([Ca²⁺]_i). Numbers adjacent to traces indicate measured peak values of [Ca²⁺]_i. (B) Time derivative of responses corresponding to traces in (A) as indicated by the arrows. Dashed vertical lines denote the delays between the flash delivery and the maximal rate of C_m increase. (C) Ca²⁺ dependence of the maximal rate of capacitance increase. The curve was obtained by fitting the data to the following function: (f F/s) = (3008 ± 792) × ([Ca²⁺]^{(5.3 ± 2.3)})/((21.9 ± 3.8 μM)^{(5.3 ± 2.3)} + [Ca²⁺]^{(5.3 ± 2.3)}). Each filled circle represents the average ± standard error of three to five measurements. (D) Ca²⁺ dependence of the delay to the maximal rate of capacitance increase. The curve was obtained by fitting the data to the following exponential regression algorithm: delay (s) = (0.55 ± 0.15 s) × (exp(−(0.057 ± 0.015 μM⁻¹) × [Ca²⁺])), where the parameters are in the format mean ± standard error. Modified with permission from Figure 2 in Kreft et al. (2004).