Bacterial endotoxin induces [Ca²⁺]i transients and changes the organization of actin in microglia

Abstract

We have employed amoeboid microglia purified from primary cultures of neonatal rat brain to examine the effect of bacterial lipopolysaccharide (LPS), a potent activator of immune cells, on intracellular calcium concentration ([Ca2+]i) in brain macrophages. In single brain macrophages loaded with indo 1, pulse administration of LPS elicited a rapid and transient increase in [Ca2+]i. From a total of 70 cells examined, all responded to LPS with a similar [Ca2+]i transient, indicating a good homogeneity of the cell population with regard to the LPS response. It was concluded that the rise of cytosolic [Ca2+]i originated from intracellular stores because the response to LPS occurred similarly in the presence or in the absence of extracellular Ca2+. A second administration of LPS to the same cells resulted in a second but reduced [Ca2+]i transient. In contrast to the first response to LPS, this second response was totally dependent on the presence of Ca2+ in the extracellular medium. The first response to LPS was strongly inhibited by ruthenium red and could be suppressed in a reversible manner by preincubating the cells with caffeine in the absence of Ca2+ in the extracellular medium. These results indicate that caffeine-sensitive intracellular Ca2+ stores may be the major source of Ca2+ in the response of brain macrophages to LPS. The possible release of Ca2+ from phosphatidylinositol(3,4,5)-trisphosphate (IP3)-sensitive stores in brain macrophages was also evaluated by stimulating cells with the IP3-mobilizing agonist histamine. Brain macrophages were heterogeneous with regard to the histamine response since histamine induced a [Ca2+]i rise in only 30% of cells examined.(ABSTRACT TRUNCATED AT 250 WORDS)

P2X4-Receptor-Mediated Synthesis and Release of Brain-Derived Neurotrophic Factor in Microglia Is Dependent on Calcium and p38-Mitogen-Activated Protein Kinase Activation

“…Similarly, neither BDNF release or accumulation were affected by treating the microglia with glutamate (100 M) (supplemental Fig. 3, available at www.jneurosci.org as supplemental material) or with lipopolysaccharide (1 g/ml), both of which are known to cause release of Ca 2ϩ from ER stores in microglia (Bader et al, 1994;D'Antoni et al, 2008). Thus, in contrast to influx of extracellular Ca 2ϩ , release of Ca 2ϩ from ER stores was insufficient to cause the release of BDNF or its accumulation in the microglia.…”

Inhibition of lipopolysaccharide‐induced cyclooxygenase‐2, tumor necrosis factor‐α and [Ca2+]i responses in human microglia by the peripheral benzodiazepine receptor ligand PK11195

“…The identification of the former pathway was based solely on block of LPS responses with cell preincubation with caffeine and not direct effects of caffeine to alter [Ca 2+ ] i ; such stores seem not generally reported in rodent cells and human microglia do not show transient changes in [Ca 2+ ] i with application of caffeine (unpublished data). It should also be noted that LPS was applied to rat microglia at 50 lg/mL (Bader et al 1994) which was 500 times the level employed in the present study.…”

“…This result could suggest that depletion from ER stores was not involved but this conclusion would require the proviso that ATP to have fully depleted ER stores. LPS responses have previously been recorded from rat microglia using a protocol with two successive applications of the endotoxin (Bader et al 1994). The initial response of a rapid, transient change in [Ca 2+ ] i was suggested to arise from a caffeine-sensitive intracellular store and the second response due to a La 3+ -sensitive influx pathway.…”

Mechanisms Underlying Neuronal Death Induced by Chromogranin A-activated Microglia

“…Amoeboid microglia isolated from neonatal rat brain cultures consist of flat, round, or spindleshaped highly adherent cells. They respond to bacterial endotoxin (32) and express Fc (33) and CR3 complement receptors (26).…”

“…The immunoprecipitates were washed twice with buffer containing 20 mM HEPES, 10% glycerol, 0.1% Triton X-100, and 500 mM NaCl followed by two washes with the same buffer containing 150 mM NaCl. Kinase activity was assayed by incubating the resuspended pellet at 30°C in the presence of 10 Ci of [␥- 32 P]ATP and the appropriate substrate: myelin basic protein (3 g) for p38 MAP kinase and glutathione S-transferase-c-Jun (1 g) for JNK1. After 20 min, the reaction was stopped by the addition of electrophoresis sample buffer and heating at 100°C for 5 min.…”