Malignant gliomas have been shown to release glutamate, which kills surrounding brain cells, creating room for tumor expansion. This glutamate release occurs primarily via system x C À , a Na + -independent cystine-glutamate exchanger. We show here, in addition, that the released glutamate acts as an essential autocrine/paracrine signal that promotes cell invasion. Specifically, chemotactic invasion and scrape motility assays each show dose-dependent inhibition of cell migration when glutamate release was inhibited using either S-(4)-CPG or sulfasalazine, both potent blockers of system x C À . This inhibition could be overcome by the addition of exogenous glutamate (100 Mmol/L) in the continued presence of the inhibitors. Migration/invasion was also inhibited when Ca 2+ -permeable A-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPA-R) were blocked using GYKI or Joro spider toxin, whereas CNQX was ineffective. Ca 2+ imaging experiments show that the released glutamate activates Ca 2+ -permeable AMPA-R and induces intracellular Ca 2+ oscillations that are essential for cell migration. Importantly, glioma cells release glutamate in sufficient quantities to activate AMPA-Rs on themselves or neighboring cells, thus acting in an autocrine and/or paracrine fashion. System x C À and the appropriate AMPA-R subunits are expressed in all glioma cell lines, patient-derived glioma cells, and acute patient biopsies investigated. Furthermore, animal studies in which human gliomas were xenographed into scid mice show that chronic inhibition of system x C À -mediated glutamate release leads to smaller and less invasive tumors compared with saline-treated controls. These data suggest that glioma invasion is effectively disrupted by inhibiting an autocrine glutamate signaling loop with a clinically approved candidate drug, sulfasalazine, already in hand.

Ca(2+)-activated K(+) (K(Ca)) channels are a unique family of ion channels because they are capable of directly communicating calcium signals to changes in cell membrane potential required for cellular processes including but not limited to cellular proliferation and migration. It is now possible to distinguish three families of K(Ca) channels based on differences in their biophysical and pharmacological properties as well as genomic sequence. Using a combination of biochemical, molecular, and biophysical approaches, we show that human tumor cells of astrocytic origin, i.e. glioma cells, express transcripts for all three family members of K(Ca) channels including BK, IK, and all three SK channel types (SK1, SK2, and SK3). The use of selective pharmacological inhibitors shows prominent expression of currents that are inhibited by the BK channel specific inhibitors iberiotoxin and paxilline. However, despite the presence of transcripts for IK and SK, neither clotrimazole, an inhibitor of IK channels, nor apamin, known to block most SK channels inhibited any current. The exclusive expression of functional BK channels was further substantiated by shRNA knockdown experiments, which selectively reduced iberiotoxin sensitive currents. Western blotting of patient biopsies with antibodies specific for all three KCa channel types further substantiated the exclusive expression of BK type KCa channels in vivo. This finding is in sharp contrast to other cancers that express primarily IK channels.

Relative contribution of chloride channels and transporters to regulatory volume decrease in human glioma cells.

Voltage-dependent large-conductance Ca 2+ -activated K + channels, often referred to as BK channels, are a unique class of ion channels coupling intracellular chemical signaling to electrical signaling. BK channel expression has been shown to be up-regulated in human glioma biopsies, and expression levels correlate positively with the malignancy grade of the tumor. Glioma BK channels (gBK) are a splice variant of the hslo gene, are characterized by enhanced sensitivity to [Ca 2+ ] i , and are the target of modulation by growth factors. By using the selective pharmacological BK channel inhibitor iberiotoxin, we examined the potential role of these channels in tumor growth. Cell survival assays examined the ability of glioma cells to grow in nominally serum-free medium. Under such conditions, BK channel inhibition by iberiotoxin caused a dose-and time-dependent decrease in cell number discernible as early as 72 hr after exposure and maximal growth inhibition after 4-5 days. FACS analysis shows that IbTX treatment arrests glioma cells in S phase of the cell cycle, whereupon cells undergo cell death. Interestingly, IbTX effects were nullified when cells were maintained in 7% fetal calf serum. Electrophysiological analysis, in conjunction with biotinylation studies, demonstrates that serum starvation caused a significant translocation of BK channel protein to the plasma membrane, corresponding to a two-to threefold increase in whole-cell conductance, but without a change in total gBK protein. Hence, expression of functional gBK channels appears to be regulated in a growth-factor-dependent manner, with enhanced surface expression promoting tumor cell growth under conditions of growth factor deprivation as might occur under in vivo conditions. Keywords BK channel; glioma; proliferation; iberiotoxin; cell growth Gliomas are primary brain tumors believed to originate from normal glial cells or their progenitors. They account for 20% of all brain malignancies and are characterized by relentless growth and aggressive invasion into the brain parenchyma. These features make surgical treatment of these cancers difficult, resulting in a dim prognosis for affected patients. Much has been learned regarding the unusual growth characteristics of gliomas. Most notably, it has been demonstrated that these tumors can grow under conditions that would be adverse to growth of nonmalignant cells (Rouzaire-Dubois et al., 1993). Thus, whereas normal cell growth depends on the continuous presence of growth factors, glioma cells have developed autocrine and paracrine signaling cascades that support their growth in the absence of exogenous growthstimulating factors (Rozengurt, 1999;Heasley, 2001;Gerber and Ferrara, 2003;Yu et al., 2003). Such factors include, for example, neuregulin-1, which is synthesized and released by , 1997) and which signals through heterodimeric erb-B2/erb-B4 receptors that are overexpressed in glioma cells (Westphal et al., 1997). In at least 50% of human gliomas, the epidermal growth factor receptor (EGF-R...

SummaryThe bacterial endospore cortex peptidoglycan is synthesized between the double membranes of the developing forespore and is required for attainment of spore dehydration and dormancy. The Bacillus subtilis spoVB, spoVD and spoVE gene products are expressed in the mother cell compartment early during sporulation and play roles in cortex synthesis. Here we show that mutations in these genes block synthesis of cortex peptidoglycan and cause accumulation of peptidoglycan precursors, indicating a defect at the earliest steps of peptidoglycan polymerization. Loss of spoIV gene products involved in activation of later, s K -dependent mother cell gene expression results in decreased synthesis of cortex peptidoglycan, even in the presence of the SpoV proteins that were synthesized earlier, apparently due to decreased precursor production. Data show that activation of s K is required for increased synthesis of the soluble peptidoglycan precursors, and Western blot analyses show that increases in the precursor synthesis enzymes MurAA, MurB, MurC and MurF are dependent on s K activation. Overall, our results indicate that a decrease in peptidoglycan precursor synthesis during early sporulation, followed by renewed precursor synthesis upon s K activation, serves as a regulatory mechanism for the timing of spore cortex synthesis.

Glioma cells prominently express a unique splice variant of a large conductance, calcium-activated potassium channel (BK channel). These channels transduce changes in intracellular calcium to changes of K ؉ conductance in the cells and have been implicated in growth control of normal and malignant cells. The Ca 2؉ increase that facilitates channel activation is thought to occur via activation of intracellular calcium release pathways or influx of calcium through Ca 2؉ -permeable ion channels. We show here that BK channel activation involves the activation of inositol 1,4,5-triphosphate receptors (IP 3 R), which localize near BK channels in specialized membrane domains called lipid rafts. Disruption of lipid rafts with methyl-␤-cyclodextrin disrupts the functional association of BK channel and calcium source resulting in a >50% reduction in K ؉ conductance mediated by BK channels. The reduction of BK current by lipid raft disruption was overcome by the global elevation of intracellular calcium through inclusion of 750 nM Ca 2؉ in the pipette solution, indicating that neither the calcium sensitivity of the channel nor their overall number was altered. Additionally, pretreatment of glioma cells with 2-aminoethoxydiphenyl borate to inhibit IP 3 Rs negated the effect of methyl-␤-cyclodextrin, providing further support that IP 3 Rs are the calcium source for BK channels. Taken together, these data suggest a privileged association of BK channels in lipid raft domains and provide evidence for a novel coupling of these Ca 2؉ -sensitive channels to their second messenger source.

Glioma cells show up-regulation and constitutive activation of erbB2, and its expression correlates positively with increased malignancy. A similar correlation has been demonstrated for the expression of gBK, a calcium-sensitive, large-conductance K + channel. We show here that glioma BK channels are a downstream target of erbB2/neuregulin signaling. Tyrphostin AG825 was able to disrupt the constituitive erbB2 activation in a dose-dependent manner, causing a 30-mV positive shift in gBK channel activation in cell-attached patches. Conversely, maximal stimulation of erbB2 with a recombinant neuregulin (NRG-1β) caused a 12-mV shift in the opposite direction. RT-PCR studies reveal no change in the BK splice variants expressed in treated glioma cells. Furthermore, isolation of surface proteins through biotinylation did not show a change in gBK channel expression, and probing with phospho-specific antibodies showed no alteration in channel phosphorylation. However, fura-II Ca 2+ fluorescence imaging revealed a 35% decrease in the free intracellular Ca 2+ concentration after erbB2 inhibition and an increase in NRG-1β-treated cells, suggesting that the observed changes most likely were due to alterations in [Ca 2+ ] i . Consistent with this conclusion, neither tyrphostin AG825 nor NRG-1β was able to modulate gBK channels under inside-out or whole-cell recording conditions when intracellular Ca 2+ was fixed. Thus, gBK channels are a downstream target for the abundantly expressed neuregulin-1 receptor erbB2 in glioma cells. However, unlike the case in other systems, this modulation appears to occur via changes in [Ca 2+ ] i without changes in channel expression or phosphorylation. The enhanced sensitivity of gBK channels in glioma cells to small, physiological Ca 2+ changes appears to be a prerequisite for this modulation.Keywords growth factor; potassium channel; brain tumor; patch clamp; neuregulin Voltage-dependent, large-conductance, calcium-activated potassium channels (BK) differ from many other ion channels in that, as the name implies, they are activated by two physiological processes; a change in voltage and changes in intracellular Ca 2+ concentration. Under physiological conditions, Ca 2+ binding is required for channel activation. BK channels are expressed in a broad spectrum of cells and tissue types and across many species. In excitable cells, BK channel expression and activity persist throughout the life of the cell. BK channels play important roles in mediating different phases of hyperpolarization and afterhyperpolarization after an action potential and are involved in neurotransmitter release and muscle relaxation (for review see Sah, 1996;Gribkoff et al., 2001;Sah and Faber, 2002;Calderone, 2002). For nonexcitable cells, the role that these channels play in cell biology is less well understood. In glial cells, the highest channel activity is observed in undifferentiated, proliferating cells, whereas channels are largely absent in differentiated astrocytes (Puro et al., 1989;Sontheimer et al., ...

ObjectiveTo design and validate a brief set of measures identifying staff and work areas exhibiting low levels of resilience within healthcare organizations.Data sources/Study designPrimary data were gathered via survey administration between April and August of 2016 from 33,622 respondents across 123 facilities. These surveys included pilot items designed to measure resilience and were administered to all employees alongside employee engagement surveys.Data collection/Extraction methodsFollowing the data collection period for the pilot survey, data from all organizations were integrated into a single analytical dataset. Factor analyses were used to determine the underlying constructs of healthcare worker resilience. Cronbach's alpha and correlation analyses tested the internal consistency and validity of the instrument.Principal findingsA brief set consisting of eight items was identified as a psychometrically validated measure of resilience. This measure consists of two subscales, Activation and Decompression. These measures exist independent of employee engagement, indicating an empirical distinction between the two concepts. Resilience was found to predict 38% of variance in engagement scores.ConclusionsAn eight-item instrument can accurately measure resilience to identify burnout risk and serve as a predictor of other workforce outcomes such as engagement.