Naloxone and Ouabain in Ultralow Concentrations Restore Na+/K+-ATPase and Cytoskeleton in Lipopolysaccharide-treated Astrocytes

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.