Ammonium triggers calcium elevation in cultured mouse microglial cells by initiating Ca²⁺ release from thapsigargin-sensitive intracellular stores

Abstract

Microglial cells are thought to serve as sensors for pathologic events in the brain. In the present study we demonstrate that these cells respond with an increase in intracellular calcium concentration ([Ca²⁺]_i) to intracellular alkaline shifts induced by either application of NH₃/NH₄ ⁺ or by an extracellular alkaline shift. The cytoplasmic pH (pH_i) and [Ca²⁺]_i in cultured mouse microglial cells were studied employing the fluorescent probes BCECF and fura-2, respectively. Application of NH₃/NH₄ ⁺ caused an initial rapid alkalinization followed by a slow recovery towards the resting level, while application of alkaline (pH 8.2) solution triggered a slower rise in pH_i. The [Ca²⁺]_i elevation triggered by NH₃/NH₄ ⁺ and extracellular alkaline shift were caused by different mechanisms: extracellular alkalinization induced a transmembrane Ca²⁺ entry, whereas NH₃/NH₄ ⁺ triggered Ca²⁺ release from thapsigargin- and ATP-sensitive intracellular pools. The mobilization of intracellular Ca²⁺ caused by NH₃/NH₄ ⁺ was blocked by a specific inhibitor of phospholipase C, U-73122, but was not affected by an inhibitor of G-protein, pertussis toxin. This implies that NH₃/NH₄ interacts with phospholipase C and leads to an increase in the intracellular level of inositol 1,4,5-trisphosphate (InsP ₃). In contrast to a previous study using a microglial cell line, application of NH₃/NH₄ ⁺ did not result in a release of tumor necrosis factor α (TNF-α), a marker of microglial activation, in the primary microglial cells. This implies that ammonium does not lead to activation of microglia in the culture model.