MG Human Astrocytoma Cells Research Articles

Generation of a Live-Attenuated Strain of Chikungunya Virus from an Indian Isolate for Vaccine Development.

Chikungunya virus (CHIKV) re-emergence in the last decade has resulted in explosive epidemics. Along with the classical symptoms of fever and debilitating arthralgia, there were occurrences of unusual clinical presentations such as neurovirulence and mortality. These generated a renewed global interest to develop prophylactic vaccines. Here, using the classical approach of virus attenuation, we developed an attenuated CHIKV strain (RGCB355/KL08-p75) for the purpose. Repeated passaging (75 times) of a local clinical isolate of ECSA lineage virus in U-87 MG human astrocytoma cells, an interferon-response-deficient cell line, resulted in efficient adaptation and attenuation. While experimental infection of 3-day old CHIKV-susceptible BALB/c pups with the parent strain RGCB355/KL08-p4 resulted in death of all the animals, there was 100% survival in mice infected with the attenuated p75. In adult, immunocompetent, CHIKV-non-susceptible C57BL/6 mice, inoculation with p75 induced high antibody response without any signs of disease. Both p4 and p75 strains are uniformly lethal to interferon-response-deficient AG129 mice. Passive protection studies in AG129 mice using immune serum against p75 resulted in complete survival. Whole-genome sequencing identified novel mutations that might be responsible for virus attenuation. Our results establish the usefulness of RGCB355/KL08-p75 as a strain for vaccine development against chikungunya.

Dithiolethiones D3T and ACDT Protect Against Ferroptosis Induced by Erastin and RSL3

Ferroptosis is an iron‐dependent and non‐apoptotic form of cell death characterized by increased lipid peroxidation. It is morphologically, biochemically, and genetically different from other types of cell death. Erastin and RSL3 are the canonical inducers of ferroptosis. While erastin inhibits the cystine/glutamate antiporter (xCT) leading to intracellular glutathione (GSH) depletion, RSL3 directly inhibits the glutathione peroxidase‐4 (GPX4) enzyme, resulting in increased lipid peroxidation and ferroptosis. Dithiolethiones 3H‐1,2‐dithiole‐3‐thione (D3T) and 5‐amino‐3‐thioxo‐3H‐(1,2) dithiole‐4‐carboxylic acid ethyl ester (ACDT) are lipophilic, organosulfur compounds that are known to activate the Nrf2 transcription factor and induce the cellular antioxidant machinery. They have previously been shown to be cytoprotective against various oxidative stressors both in vitro and in vivo. Furthermore, as lipophilic antioxidants are known to oppose ferroptosis and the activation of Nrf2 has been reported to inhibit this form of cell death, the present study was aimed to evaluate the protective effect of dithiolethiones against erastin‐ and RSL3‐induced ferroptosis in U‐87 MG human astrocytoma cells. Ferrostatin‐1, a known ferroptosis inhibitor was used as a standard comparator. Cells were pretreated with either 50 µM D3T, ACDT, or 10 µM ferrostatin‐1 for 24 hours followed by another 24 hour exposure to either 20 µM erastin or 10 µM RSL3. Pretreatment with dithiolethiones reversed erastin‐induced cell death measured by MTS cell viability assay and upregulated xCT transporter expression indicated by western blotting. Dithiolethiones increased intracellular GSH (GSH‐Glo luminescence assay) and alleviated lipid peroxidation (TBARS assay) against erastin. RSL3‐induced ferroptosis and lipid peroxidation were also counteracted by both the dithiolethiones, however no effect was observed on GPX4 enzyme levels. These effects of dithiolethiones were comparable to those of the standard ferroptosis inhibitor ferrostatin‐1. Collectively, our data indicate a strong ability of dithiolethiones to counteract ferroptosis, and is the first such study in astrocytes. D3T and ACDT can therefore be potential therapeutic candidates in the treatment of ferroptosis‐related brain disorders.

Intracellular nickel accumulation induces apoptosis and cell cycle arrest in human astrocytic cells.

Nickel, a heavy metal found in electronic wastes and fume from electronic cigarettes, induces neuronal cell death and is associated with neurocognitive impairment. Astrocytes are the first line of defense against nickel after entering the brain; however, the effects of nickel on astrocytes remain unknown. Herein, we investigated the effect of nickel exposure on cell survival and proliferation and the underlying mechanisms in U-87 MG human astrocytoma cells and primary human astrocytes. Intracellular nickel levels were elevated in U-87 MG cells in a dose- and time-dependent manner after exposure to nickel chloride. The median toxic concentrations of nickel in astrocytoma cells and primary human astrocytes were 600.60 and >1000µMat 48h post-exposure, respectively. Nickel exposure triggered apoptosis in concomitant with the decreased expression of anti-apoptotic B-cell lymphoma protein (Bcl-2) and increased caspase-3/7 activity. Nickel induced reactive oxygen species formation. Additionally, nickel suppressed astrocyte proliferation in a dose- and time-dependent manner by delaying G2 to M phase transition through the upregulation of cyclin B1 and p27 protein expression. These results indicate that nickel-induced cytotoxicity of astrocytes is mediated by the activation of apoptotic pathway and disruption of cell cycle regulation.

Extracellular cardiolipin modulates glial phagocytosis and cytokine secretion in a toll‐like receptor 4‐dependent manner

AbstractBackgroundCardiolipin (CL) is a mitochondrial membrane phospholipid that acts as a signaling molecule when released into the extracellular space from degenerating and dying brain cells. Previous research has shown that extracellular CL inhibits secretion of pro‐inflammatory mediators by immune‐activated microglia. CL alone also increases the phagocytic activity of microglia and upregulates their expression of brain‐derived neurotrophic factor (BDNF) and glial cell‐derived neurotrophic factor (GDNF). The mechanisms by which CL modulates microglia functions, in a manner that is potentially beneficial in Alzheimer’s disease, are unknown. Furthermore, the effects of CL on astrocytes have not been explored. Since microglia and astrocytes express toll‐like receptor (TLR) 4, we hypothesized that extracellular CL interacts with this receptor, modulating immune functions of glia.MethodWe studied the effects of CL applied to the culture media on select functions of primary murine microglia and astrocytes, BV‐2 murine microglia, U118 MG human astrocytoma cells, and THP‐1 human microglia‐like cells. Secretion of inflammatory cytokines was monitored by enzyme‐linked immunosorbent assay (ELISA) or immunoblotting. The phagocytic activity of primary microglia and astrocytes was measured with fluorescent latex microspheres. The internalization of the microspheres was verified using confocal microscopy.ResultExtracellular CL (0.4‐14 μM) alone upregulates the phagocytic activity and release of monocyte chemoattractant protein (MCP)‐1 by murine microglia and human astrocytic cells. TLR 4‐specific antagonist TAK‐242 and blocking antibodies inhibit the upregulated phagocytic activity and MCP‐1 secretion by CL‐treated microglia and human THP‐1 microglia‐like cells. CL increases the secretion of interferon (IFN) γ‐induced protein (IP)‐10 and IFN‐β by THP‐1 microglia‐like cells and human U118 MG astrocytoma cells, respectively. CL inhibits the secretion of cytotoxins by several types of activated microglia and astrocytes. Amyloid β (Aβ42) peptide‐ and lipopolysaccharide‐induced secretion of cytokines by primary murine microglia and U118 MG astrocytoma cells, respectively, is reduced by extracellular CL.ConclusionExtracellular CL can interact with TLR 4 to modulate select functions of microglia and astrocytes. Importantly, we demonstrate that CL inhibits amyloid‐β peptide‐induced secretion of inflammatory cytokines by microglia and reduces glia‐mediated neuron death. Therefore, CL or liposomes containing CL could be developed as potential therapeutic agents for Alzheimer’s disease.

Anti‑inflammatory actions of gabapentin and pregabalin on the substance P‑induced mitogen‑activated protein kinase activation in U373 MG human glioblastoma astrocytoma cells.

Gabapentin (GBP) and pregabalin (PGB) exert antinociceptive effects on chronic nociceptive responses with neuropathic or inflammatory conditions. Furthermore, it is considered that GBP and PGB exhibit anti‑inflammatory effects by modulating the substance P (SP)‑mediated neurokinin‑1 receptor (NK1R; a SP receptor) response. Thus, in the present study, the effects of GBP and PGB on SP‑induced activation were investigated in the human glioblastoma astrocytoma U373 MG cell line, which expresses high levels of functional high‑affinity NK1R, and produces interleukin (IL)‑6 and IL‑8 in response to SP. The results indicated that GBP and PGB suppressed the SP‑induced production of IL‑6, and IL‑8 in U373 MG cells. Furthermore, GBP and PGB inhibited the SP‑induced phosphorylation of p38 mitogen‑activated protein kinase (MAPK) and nuclear factor (NF)‑κB, and the nuclear translocation of NF‑κB in U373 MG cells. Together, these observations suggest that GBP and PGB likely prevent SP‑induced IL‑6 and IL‑8 production in U373 MG cells via the inhibition of signaling molecules, including p38 MAPK and NF‑κB, thereby exhibiting antineuroinflammatory effects.

Cadmium-induced IL-6 and IL-8 expression and release from astrocytes are mediated by MAPK and NF-κB pathways

Chronic exposure to cadmium has been linked to brain cancers, learning disabilities and memory deficits. Previous studies of cadmium toxicity in the central nervous system report cadmium induces oxidative stress in neurons and astrocytes. In the peripheral system, cadmium promotes interleukin-6 (IL-6) and IL-8 production and release. Elevation of IL-6 expression is linked to the pathogenesis of neurodegenerative diseases and astrogliosis. IL-8 plays a role in angiogenesis of gliomas and neurodegenerative diseases. Herein, the effects of non-toxic concentrations of cadmium on the production of IL-6 and IL-8 and the underlying mechanisms were investigated. U-87 MG human astrocytoma cells and primary human astrocytes were exposed to cadmium chloride. At 24h post-exposure to 1 and 10μM, levels of intracellular cadmium in U-87 MG cells were 11.89±3.59 and 53.08±7.59μg/g wet weight, respectively. These concentrations had minimal effects on cell morphology and viability. IL-6 and IL-8 mRNA levels and secretion increased in dose- and time-dependent manners post cadmium exposure. Acute exposure to cadmium increased phosphorylation of ERK1/2, p38 MAPK, and p65 NF-κB. Pretreatment with U0126–an inhibitor of MEK1 and MEK2 kinases–SB203580–a p38 MAPK inhibitor–and SC-514–an IKKβ inhibitor–suppressed cadmium-induced IL-8 expression and release. Upregulation of cadmium-induced IL-6 was inhibited by U0126 and SC-514, but not SB203580. On the other hand, SP600125–a JNK inhibitor–and celecoxib–a selective COX-2 inhibitor–had no effect on production of both cytokines. In conclusion, non-toxic concentrations of cadmium can stimulate IL-6 and IL-8 release through MAPK phosphorylation and NF-κB activation. Suppressing IL-6 and IL-8 production could be novel approaches to prevent cadmium-induced angiogenesis in gliomas and inflammation in the brain.

Interleukin-1 beta-induced up-regulation of opioid receptors in the untreated and morphine-desensitized U87 MG human astrocytoma cells

BackgroundInterleukin-1beta (IL-1β) is a pro-inflammatory cytokine that can be produced in the central nervous system during inflammatory conditions. We have previously shown that IL-1β expression is altered in the rat brain during a morphine tolerant state, indicating that this cytokine may serve as a convergent point between the immune challenge and opiate mediated biological pathways. We hypothesized that IL-1β up-regulates opioid receptors in human astrocytes in both untreated and morphine-desensitized states.MethodsTo test this hypothesis, we compared the basal expression of the mu (MOR), delta (DOR), and kappa (KOR) opioid receptors in the human U87 MG astrocytic cell line to SH-SY5Y neuronal and HL-60 immune cells using absolute quantitative real time RT-PCR (AQ-rt-RT-PCR). To demonstrate that IL-1β induced up-regulation of the MOR, DOR and KOR, U87 MG cells (2 x 105 cells/well) were treated with IL-1β (20 ng/mL or 40 ng/mL), followed by co-treatment with interleukin-1 receptor antagonist protein (IL-1RAP) (400 ng/mL or 400 ng/mL). The above experiment was repeated in the cells desensitized with morphine, where U87 MG cells were pre-treated with 100 nM morphine. The functionality of the MOR in U87 MG cells was then demonstrated using morphine inhibition of forksolin-induced intracellular cAMP, as determined by radioimmunoassay.ResultsU87 MG cells treated with IL-1β for 12 h showed a significant up-regulation of MOR and KOR. DOR expression was also elevated, although not significantly. Treatment with IL-1β also showed a significant up-regulation of the MOR in U87 MG cells desensitized with morphine. Co-treatment with IL-1β and interleukin-1 receptor antagonist protein (IL-1RAP) resulted in a significant decrease in IL-1β-mediated MOR up-regulation.ConclusionOur results indicate that the pro-inflammatory cytokine, IL-1β, affects opiate-dependent pathways by up-regulating the expression of the MOR in both untreated and morphine-desensitized U87 MG.

Ocular Manifestations and Treatment Considerations for the Patient With Cardio-Facial-Cutaneous Syndrome

Chronic exposure to cadmium has been linked to brain cancers, learning disabilities and memory deficits. Previous studies of cadmium toxicity in the central nervous system report cadmium induces oxidative stress in neurons and astrocytes. In the peripheral system, cadmium promotes interleukin-6 (IL-6) and IL-8 production and release. Elevation of IL-6 expression is linked to the pathogenesis of neurodegenerative diseases and astrogliosis. IL-8 plays a role in angiogenesis of gliomas and neurodegenerative diseases. Herein, the effects of non-toxic concentrations of cadmium on the production of IL-6 and IL-8 and the underlying mechanisms were investigated. U-87 MG human astrocytoma cells and primary human astrocytes were exposed to cadmium chloride. At 24 h post-exposure to 1 and 10 μM, levels of intracellular cadmium in U-87 MG cells were 11.89 ± 3.59 and 53.08 ± 7.59 μg/g wet weight, respectively. These concentrations had minimal effects on cell morphology and viability. IL-6 and IL-8 mRNA levels and secretion increased in dose- and time-dependent manners post cadmium exposure. Acute exposure to cadmium increased phosphorylation of ERK1/2, p38 MAPK, and p65 NF-κB. Pretreatment with U0126–an inhibitor of MEK1 and MEK2 kinases–SB203580–a p38 MAPK inhibitor–and SC-514–an IKKβ inhibitor–suppressed cadmium-induced IL-8 expression and release. Upregulation of cadmium-induced IL-6 was inhibited by U0126 and SC-514, but not SB203580. On the other hand, SP600125–a JNK inhibitor–and celecoxib–a selective COX-2 inhibitor–had no effect on production of both cytokines. In conclusion, non-toxic concentrations of cadmium can stimulate IL-6 and IL-8 release through MAPK phosphorylation and NF-κB activation. Suppressing IL-6 and IL-8 production could be novel approaches to prevent cadmium-induced angiogenesis in gliomas and inflammation in the brain.

Substance P receptor in U373 MG human astrocytoma cells activates mitogen-activated protein kinases ERK1/2 through Src

Substance P (SP) acting through substance P receptor (SPR) increases the proliferation of glioblastoma cells. At the molecular level, stimulation of SPR in human U373 MG glioblastoma cells results in phosphorylation of mitogen-activated protein kinases ERK1/2. Examination of the underlying mechanism reveals that SPR mediates ERK1/2 phosphorylation in a calcium-dependent manner. Surprisingly, blockade of epidermal growth factor receptor (EGFR), which is transactivated by SPR, has a minimal effect on SPR-mediated ERK1/2 phosphorylation. However, SPR-mediated ERK1/2 phosphorylation is significantly reduced by the Src kinase inhibitor PP2. Interestingly, ERK1/2 in U373 MG cells is also activated by several other mitogenic G protein-coupled receptors (GPCRs) including alpha(1B)-adrenergic, M(3)-muscarinic, and H(1)-histaminergic in an Src-dependent manner. We conclude that c-Src is a mediator of SP-stimulated ERK1/2 phosphorylation in human U373 MG glioblastoma cells.

Mechanism of extracellular signal-regulated kinase activation by the CB(1) cannabinoid receptor.

Cannabinoids, the active components of marijuana and their endogenous counterparts, exert many of their actions in brain through the seven-transmembrane receptor CB(1). This receptor is coupled to the activation of the extracellular signal-regulated kinase (ERK) cascade. However, the precise molecular mechanism for CB(1)-mediated ERK activation is still unknown. Here, we show that in U373 MG human astrocytoma cells, CB(1) receptor activation with the cannabinoid agonist delta(8)-tetrahydrocannabinol dimethyl heptyl (HU-210) was coupled to ERK activation and protection from ceramide-induced apoptosis. HU-210-induced ERK activation was inhibited by tyrphostin AG1478 and PP2, widely employed inhibitors of the epidermal growth factor receptor (EGF(R)) and the Src family of cytosolic tyrosine kinases, respectively. However, HU-210 stimulation resulted in neither EGF(R) phosphorylation, Src tyrosine phosphorylation, nor increased Src activity. In addition, dominant-negative forms of both proteins were unable to prevent cannabinoid-induced ERK activation, thus excluding the existence of CB(1)-mediated EGF(R) transactivation or Src activation. Wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294,002), inhibitors of the phosphatidylinositol 3-kinase (PI3K) signaling pathway, blocked cannabinoid-induced ERK activation. Likewise, HU-210 stimulated the PI3K downstream targets protein kinase B (PKB), as shown by its phosphorylation in Thr 308 and Ser 473 residues, and Raf-1. Moreover, betagamma subunit release mimicked ERK and PI3K/PKB activation, suggesting that activation of class IB PI3K mediates cannabinoid action. Pro-survival HU-210 action also required activation of both PI3K and ERK signaling pathways. In conclusion, CB(1)-induced ERK activation was mediated by PI3K(IB) and this effect may have important consequences in the control of cell death/survival decision.

Intracellular Ca2+ mediates lipoxygenase-induced proliferation of U-373 MG human astrocytoma cells.

The role of intracellular Ca2+ in the regulation of tumor cell proliferation by products of arachidonic acid (AA) metabolism was investigated using U-373 MG human astrocytoma cells. Treatment with nordihydroguaiaretic acid (NDGA), a lipoxygenase (LOX) inhibitor, or caffeic acid (CA), a specific 5-LOX inhibitor, suppressed proliferation of the tumor cells in a dose-dependent manner. However, indomethacin (Indo), a cyclooxygenase (COX) inhibitor, did not significantly alter proliferation of the tumor cells. At anti-proliferative concentrations, NDGA and CA significantly inhibited intracellular Ca2+ release induced by carbachol, a known intracellular Ca2+ agonist in the tumor cells. Exogenous administration of leukotriene B4 (LTB4), an AA metabolite of LOX pathway, enhanced proliferation of the tumor cells in a concentration-dependent fashion. In addition, LTB4 induced intracellular Ca2+ release. Intracellular Ca2+ inhibitors, such as an intracellular Ca2+ chelator (BAPTA) and intracellular Ca(2+)-release inhibitors (dantrolene and TMB-8), significantly blocked the LTB4-induced enhancement of cell proliferation and intracellular Ca2+ release. These results suggest that LOX activity may be critical for cell proliferation of the human astrocytoma cells and that intracellular Ca2+ may play a major role in the mechanism of action of LOX.

Enhancement of methylene blue-induced cytotoxicity in human brain tumor cells by an iron chelator, deferoxamine

Previously, we have reported that methylene blue (MB) induces cytotoxicity in human brain tumor cells through the generation of free radicals. In this study the effect of deferoxamine (DFO), an iron chelator, on MB-induced cytotoxicity was investigated using SK-N-MC human neuroblastoma and U-373 MG human astrocytoma cells as model cellular systems. The cytotoxic effect of MB was potentiated by DFO. The potentiation effect of DFO was significantly blocked by either stoichiometric amounts of ferric ion, various antioxidants, hydroxyl radical scavengers or intracellular Ca2+ release blockers. These results suggest that hydroxyl radical DFO. These results further suggest that the combined treatment with MB and DFO may be useful for the therapeutical applications of human brain tumors.

Cyclosporin A is a substance P (tachykinin NK 1) receptor antagonist

The immunosuppressive cyclic undecapeptide, cyclosporin A, inhibited the binding of [ 125I]substance P to tachykinin NK 1 receptors expressed by human IM-9 lymphoblastoid cells, U-373 MG human astrocytoma cells and guinea pig lung parenchyma with IC 50 values of 425 ± 58, 783 ± 180, and 784 ± 163 nM respectively. The dihydro derivative of cyclosporin A (dihydro-cyclosporin A) was an equally effective inhibitor, but the O-acetylated derivative (cyclosporin A-OAc) was 3–4-fold less potent. The cyclosporin compounds also inhibited [ 125I]neurokinin A binding to human NK 2 receptors with potencies slightly less than at NK 1 sites. In contrast, they were 8–20-fold less effective inhibitors of [ 125I]MePhe 7-neurokinin B binding to guinea pig NK 3 receptors ( p < 0.001). Thus, the cyclosporin A compounds showed selectivity for NK 1 and NK 2 receptors The structure-activity pattern for the effects of cyclosporin A compounds at tachykinin receptors differs from the pattern previously described for their immunosuppressive activity. All three compounds inhibited substance P induced interleukin-6 (IL-6) secretion from U-373 MG astrocytoma cells with potencies similar to their NK 1 receptor binding affinities. In addition, cyclosporin A blocked substance P induced phosphatidylinositol (PI) turnover in U-373 MG cells without blocking the corresponding response to histamine. This novel pharmacological profile of the cyclosporin A compounds as NK 1 receptor antagonists does not appear to correlate with other known in vitro cyclosporin A functions.

Methylene blue induces cytotoxicity in human brain tumor cells.

Methylene blue (MB), a known inhibitor of guanylyl cyclase, induced cytotoxicity in SK-N-MC human neuroblastoma and U-373 MG human astrocytoma cells in a dose-dependent manner. MB did not significantly alter cellular levels of cGMP in both cells. 8-Br cGMP, a membrane-permeable analogue of cGMP, did not decrease MB-induced cytotoxicity, indicating that cGMP may not be a major target of the cytotoxic action of MB. However, hydroxyl radical scavengers or intracellular Ca2+ modulators effectively blocked the MB-induced cytotoxicity. These results suggest that hydroxyl radical and intracellular Ca2+ may have an important involvement in the cytotoxic action of MB. These results further suggest that the treatment with MB may be useful for the therapeutic applications of human brain tumors.

Intracellular Ca2+ mediates the cytotoxicity induced by bepridil and benzamil in human brain tumor cells.

The effects of bepridil and benzamil, known Na(+)-Ca2+ exchange blockers, on the growth of human brain tumor cells were evaluated using SK-N-MC human neuroblastoma and U-373 MG human astrocytoma cells as model cellular systems. These drugs induced cytotoxicity in both cells in a dose-dependent manner. Agonist (2% fetal bovine serum) alone induced a rapid increased intracellular Ca2+ concentration and then it returned to the basal level. However, the pretreatments of these drugs resulted in a more sustained high intracellular Ca2+ concentration mobilized by an agonist. Moreover, BAPTA/AM, an intracellular Ca2+ chelator, significantly blocked the cytotoxicity induced by these drugs. These results suggest that bepridil and benzamil act as effective inhibitors of in vitro growth of human brain tumor cells and that intracellular Ca2+ may be involved in the mechanism of actions of these agents.

Potentiation of anti-proliferative effect of nitroprusside by ascorbate in human brain tumor cells

The interactions of nitric oxide (NO) and ascorbate were explored on the control of growth of human brain tumor cells. Sodium nitroprusside, a NO-generating agent, inhibited the growth of SK-N-MC human neuroblastoma cells in a dose-dependent manner. The growth inhibitory effect of nitroprusside was potentiated by sodium ascorbate and inhibited by hemoglobin. Ascorbate-induced potentiation was also observed in U-373 MG human astrocytoma cells. In both tumor cell lines, this potentiation was blocked by catalase, suggesting that hydrogen peroxide may be involved in the potentiation mechanism. In astrocytoma cells, mannitol or deferoxamine also reversed ascorbate-induced potentiation, indicating involvement of hydroxyl radical. These results suggest that the combined treatment with nitroprusside and ascorbate may be a valuable therapeutic strategy for brain tumors.

Different susceptibility to neurokinin 1 receptor antagonists of substance P and septide-induced interleukin-6 release from U373 MG human astrocytoma cell line

In a human astrocytoma cell line U373 MG, the activation of the neurokinin 1 (NK1) receptor by substance P (SP) increase, in a concentration-related manner (1 nM to 10 μM), the basal release of interleukin-6 (IL-6) as assayed by an ELISA method, in cell supernatants after 18 h of incubation. Septide, a selective NK1 receptor agonist, is equipotent to SP in inducing the IL-6 release showing similar E max (2644 ± 285 and 2830 ± 271 pg/ml) and EC 50 (15.6 ± 3.6 and 13.8 ± 3.2 nM). However, in binding assays on intact cells, septide was an about 50-fold weaker displacer of the binding of [ 3H][Sar 9,Met(O 2) 11]SP than SP ( K i's were 0.28 ± 0.1 nM and 14.2 ± 5.0 nM for SP and septide, respectively). NK2- and NK3-selective agonists (up to 1 μM) had no binding or functional effect. Highly selective non-peptide (CP96,345) or peptide (GR 82,334) NK1 receptor antagonists were more effective in antagonizing septide- (IC 50's 0.2 ± 0.06 nM and 70 ± 18 nM) than SP- (IC 50's 6.7 ± 1.3 nM and 1.95 ± 0.4 μM) induced IL-6 secretion. These data support the existence, also in human U373 MG cells, of a septide-sensitive NK1 receptor subtype(s) and/or epitope(s) blocked with high affinity by NK1 antagonist.

Evidence for the existence of ‘septide’ sensitive NK receptors in U373 MG human astrocytoma cell line

Evidence for the existence of ‘septide’ sensitive NK receptors in U373 MG human astrocytoma cell line

Calcium‐dependence of histamine‐ and carbachol‐induced inositol phosphate formation in human U373 MG astrocytoma cells: comparison with HeLa cells and brain slices

1. Histamine (1 mM) induced an accumulation of inositol monophosphate ([3H]-IP1) in the U373 MG human astrocytoma cell line which increased with time in the presence of 30 mM Li+. After a 30 min incubation period with 1 mM histamine [3H]-IP1 was the major product detected (84 +/- 1% of total [3H]-IPx) and was present at a level 11 (+/- 1) fold of basal accumulation. 2. Concentration-response curves for histamine-induced [3H]-IP1 accumulation in U373 MG cells (EC50 5.4 +/- 0.5 microM) were shifted to the right in a parallel fashion by mepyramine (slope of a Schild plot 0.99 +/- 0.08), yielding a Kd for mepyramine of 3.5 +/- 0.3 nM, consistent with the involvement of histamine H1-receptors. 3. The temelastine-sensitive binding of [3H]-mepyramine to a membrane fraction from U373 MG cells was hyperbolic and had a mean Kd of 2.5 +/- 1.0 nM. The maximum amount of temelastine-sensitive binding was 86 +/- 19 pmol g-1 membrane protein. 4. Carbachol also induced [3H]-IP1 accumulation in U373 MG cells, 2.8 (+/- 0.1) fold of basal with 1 mM carbachol, with an EC50 of 48 +/- 8 microM. Pirenzepine shifted carbachol concentration-response curves to the right (slope of Schild plot 0.89 +/- 0.07) giving a Kd for pirenzepine of 0.10 +/- 0.01 microM, suggesting that phosphoinositide hydrolysis in U373 MG cells is mediated by the M3-, rather than the M1-, muscarinic receptor subtype. 5. [3H]-IP1 accumulation induced by both 1 mM histamine and by 1 mM carbachol increased when the Ca2+ concentration of the medium was increased from 'zero' (no added Ca2+) to 0.3 mM. Histamine-stimulated [3H]-IP1 accumulation was further increased, although not so markedly, as the Ca2+ was raised to 4 mM. The same pattern was apparent with histamine-induced accumulations of [3H]-IP2 and [3H]-IP3. In contrast, [3H]-IPx accumulation in response to carbachol increased between 0.3 and 1.3 mM, but thereafter remained unchanged ([3H]-IP1) or declined ([3H]-IP2 and [3H]-IP3). 6. In HeLa cells, [3H]-IP1 accumulations induced by 1 mM histamine and 1 mM carbachol showed the same pattern of Ca2+ dependence and were independent of extracellular Ca2+ above 0.3 mM (histamine) or 1.3 mM (carbachol). The response to carbachol appeared to be mediated by an M3-muscarinic receptor (apparent Kd for pirenzepine 0.09 microM). 7. In cross-chopped slices of guinea-pig cerebral cortex and guinea-pig cerebellum, [3H]-IPI accumulation induced by 1 mM histamine in the presence of 10 mM Li+ increased as the extracellular Ca2+ was increased from 0.3 to 2.5 mM, but a further increase to 4 mM had no further effect. In contrast the response to histamine in rat cerebral cortex increased markedly between 1.3 and 4 mM Ca2+. Accumulations of [3H]-IP1 induced by carbachol in guinea-pig or rat cerebral cortical slices were not increased as extracellular Ca2+ was raised from 0.3 to 4 mM.8. Nimodipine (100 nM) and w-conotoxin (3 microM) had no significant effect on histamine-induced [3H]-IP1accumulation in rat cerebral cortical slices or in U373 MG cells. 9. We conclude that histamine-induced [3H]-IP1 accumulation in U373 MG cells does appear to have a component dependent on the extracellular Ca2+ concentration. The degree of Ca2+-dependence approaches that observed in guinea-pig cerebral cortex but is much less than in rat cerebral cortex.Whether U373 MG cells will be of use as a model system for the apparent Ca2+-entry component observed in guinea-pig or rat brain slices remains to be established.

Differential effects of methionine enkephalin on the growth of brain tumor cells.

The effect of methionine enkephalin on the growth of human brain tumor cells was investigated. The results show that this endogenous opioid has dual effects on tumor cell growth. This peptide exerted an inhibitory effect in SK-N-MC human neuroblastoma cell line; in contrast, in U-373 MG human astrocytoma cell line, the peptide showed a stimulatory effect. Treatment with naltrexone, an opioid receptor antagonist, also resulted in a similar alteration of tumor cell growth implicating that its action may be unrelated to opioid receptor blockade. These results suggest that in these tumor cell lines endogenous opioid systems may be involved in cell proliferation. Furthermore, these tumor cell lines may be useful model systems for the study of the signal transduction mechanisms of endogenous opioids.