Abstract
Astrocytes respond to inflammatory stimuli and may be important modulators of the inflammatory response in the nervous system. This study aimed first to assess how astrocytes in primary culture behave in response to inflammatory stimuli concerning intracellular Ca(2+) responses, expression of Toll-like receptor 4 (TLR4), Na(+)/K(+)-ATPase, actin filament organization, and expression of cytokines. In a cell culture model with lipopolysaccharide (LPS), astrocyte response was assessed first in the acute phase and then after incubation with LPS for 1-48 h. The concentration curve for LPS-stimulated Ca(2+) responses was bell-shaped, and the astrocytes expressed TLR4, which detects LPS and evokes intracellular Ca(2+) transients. After a long incubation with LPS, TLR4 was up-regulated, LPS-evoked Ca(2+) transients were expressed as oscillations, Na(+)/K(+)-ATPase was down-regulated, and the actin filaments were disorganized. Interleukin-1β (IL-1β) release was increased after 24 h in LPS. A second aim was to try to restore the LPS-induced changes in astrocytes with substances that may have dose-dependent anti-inflammatory properties. Naloxone and ouabain were tested separately in ultralow or high concentrations. Both substances evoked intracellular Ca(2+) transients for all of the concentrations from 10(-15) up to 10(-4) M. Neither substance blocked the TLR4-evoked Ca(2+) responses. Naloxone and ouabain prevented the LPS-induced down-regulation of Na(+)/K(+)-ATPase and restored the actin filaments. Ouabain, in addition, reduced the IL-1β release from reactive astrocytes. Notably, ultralow concentrations (10(-12) M) of naloxone and ouabain showed these qualities. Ouabain seems to be more potent in these effects of the two tested substances.
Highlights
Sion of Toll-like receptor 4 (TLR4) is up-regulated in astrocytes under neuroinflammatory conditions [3]
Activation of TLR4 leads to activation of nuclear factor-B, a factor that regulates expression of genes involved in immune responses, including release of the proinflammatory cytokines tumor necrosis factor-␣ (TNF-␣) and interleukin-1 (IL-1) [2]
Stimulation with LPS—Astrocytes were stimulated with LPS (0.01–100 ng/ml) and Ca2ϩ imaging experiments were performed on 15–17-day-old co-cultured astrocytes
Summary
Sion of TLR4 is up-regulated in astrocytes under neuroinflammatory conditions [3]. Activation of TLR4 leads to activation of nuclear factor-B, a factor that regulates expression of genes involved in immune responses, including release of the proinflammatory cytokines tumor necrosis factor-␣ (TNF-␣) and interleukin-1 (IL-1) [2]. Astrocytes express almost the same repertoire of receptors and ion channels as neurons They regulate synaptic transmission via a bidirectional communication with neurons, and they release gliotransmitters as well as factors, including cytokines, fatty acid metabolites, and free radicals (6 – 8). The Ca2ϩ signaling in astrocytes over long distances is analogous to, but much slower than, the propagation of action potentials in neurons [9] These astrocytic Ca2ϩ waves [10, 11] can be evoked by transmitters released from neurons and glial cells, followed by activation of especially G protein-coupled receptors. The Naϩ-Ca2ϩ exchanger, a Ca2ϩ transporter that controls the intracellular Ca2ϩ concentrations, is driven by the Naϩ electrochemical gradient across the PM This Naϩ pump, Naϩ/KϩATPase, indirectly modulates Ca2ϩ signaling [13], and inflammatory stimuli influence Ca2ϩ homeostasis in the astrocyte networks (5, 14 –16). We further aimed to test ouabain in low and high concentrations and its effects of Naϩ/KϩATPase activity [28] and evoked Ca2ϩ transients in astrocytes (28 –30)
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