High Extracellular Potassium Modulates Nitric Oxide Synthase Expression in Human Astrocytes

Abstract

Inducible nitric oxide synthase (iNOS) is a molecule of great interest, given the numerous biological activities of nitric oxide and the documented expression of iNOS in several CNS pathologies. There also appears to be species-dependent regulation of iNOS expression as well as CNS-specific regulation. In this study, we have examined cultures of cytokineactivated primary human astrocytes as a model system with which to study the mechanisms of iNOS regulation in human CNS. As one of the major functions of astrocytes is spatial buffering of K ϩ ion, we examined the effect of high extracellular KCl on astrocyte iNOS expression. The results demonstrate that KCl at 25-75 mM potently inhibits astrocyte nitrite production stimulated by interleukin-1 (IL-1)/interferon-␥ (IFN␥). In addition, several potassium channel inhibitors such as CsCl, tetraethylammonium, and 4-aminopyridine as well as nigericin inhibited astrocyte iNOS expression induced by IL-1/IFN␥. These results demonstrate a novel role for astrocyte potassium channel activity in modulation of astrocyte function. They further suggest neural-specific mechanisms for glial iNOS regulation.

Interleukin-1-induced changes in the glioblastoma secretome suggest its role in tumor progression

“…Recombinant human IL-1β was purchased from Peprotech (Rocky Hill, NJ). For cytokine stimulation, the cells were grown in 25 cm dishes until ~90% confluence, then incubated with IL-1β (10 ng/ml) for 15 min, which we previously determined to be sufficient to activate human astrocytes [20]. Cultures were then washed extensively with PBS to remove carry over cytokines and were further incubated with serum-free DMEM for an additional 24 h. Control medium was produced similarly except that the cytokine treatment was omitted.…”

Peptidomic Analyses of Mouse Astrocytic Cell Lines and Rat Primary Cultured Astrocytes

“…The study of stimulation-induced peptide release from astrocytes is of particular interest because peptides released in a physiologically dependent manner are more likely to be involved in cell–cell signaling. A variety of chemical stimuli known to trigger a Ca 2+ increase in astrocytes were applied to the astrocyte cell line cultures: ionomycin, ,, KCl, − serotonin, − and bradykinin. ,, By comparing the peptide content of astrocyte-conditioned media with and without chemical stimulation, we were able to study the stimulation-dependent release of peptides.…”

“…Therefore, a total of six controls were performed. The stimulus concentrations were selected according to previous studies on astrocyte secretion. − ,− Bradykinin ( m / z 1060.57) concentrations were varied to determine an optimal value large enough to induce a peptide profile change, while in sufficiently low concentrations to prevent interference with peptide detection. The stimulation experiments were performed when the cell line cultures reached 80% confluence.…”

“…These included ionomycin, 22, 36, 37 KCl, 38-40 serotonin, 41-43 and bradykinin, 21, 23, 44 all of which have been reported to evoke a Ca 2+ increase in astrocytes. Complex peptide profiles were observed after stimulation, suggesting that astrocytes are actively involved in peptide secretion.…”

“…To determine the range of peptides secreted from the astrocytes, different chemical stimuli were used to induce peptide secretion. These included ionomycin, ,, KCl, − serotonin, − and bradykinin, ,, all of which have been reported to evoke a Ca 2+ increase in astrocytes. Complex peptide profiles were observed after stimulation, suggesting that astrocytes are actively involved in peptide secretion.…”