Novel form of LTD induced by transient, partial inhibition of the Na,K-pump in rat hippocampal CA1 cells

We tested the hypothesis that transient, partial inhibition of the Na,K-pumps could produce lasting effects on synaptic efficacy in brain tissue by applying a low concentration of the ouabain analogue, dihydroouabain (DHO), to hippocampal slices for 15 min and studying the effects on field excitatory postsynaptic potentials (fEPSPs). DHO caused a suppression of fEPSPs during the application period, but this recovered only partially, to approximately 80% of control levels, after washout lasting as long as 2 h. The lasting suppression had several properties in common with low-frequency stimulation induced long-term depression (LFS-LTD), including an ability to depotentiate long-term potentiated responses. However, DHO-LTD was insensitive to blockade of N-methyl-d-aspartate or mGlu receptors or to inhibitors of protein kinase C or p38 MAP kinase. DHO-LTD did not co-occlude with LFS-LTD and therefore appears to represent a novel form of LTD. Interestingly, DHO-LTD could be prevented by pretreating slices with iberiotoxin, the selective blocker of large, Ca(2+)-dependent K+ channels ("big K," BK channels), although this toxin did not affect basal fEPSPs. Certain pathological conditions, including hypoxia and ischemia, are associated with a decrease in Na,K-pump activity and hence DHO-LTD may serve as a model for the effects on neuronal function in these conditions.     

Induction of hippocampal LTD requires nitric-oxide-stimulated PKG activity and Ca2+ release from cyclic ADP-ribose-sensitive stores.

Long-term depression (LTD) of synaptic transmission can be induced by several mechanisms, one thought to involve Ca2+-dependent activation of postsynaptic nitric oxide (NO) synthase and subsequent diffusion of NO to the presynaptic terminal. We used the stable NO donor S-nitroso-N-acetylpenicillamine (SNAP) to study the NO-dependent form of LTD at Schaffer collateral-CA1 synapses in vitro. SNAP (100 microM) enhanced the induction of LTD via a cascade that was blocked by the N-methyl-D-aspartate receptor antagonist D-2-amino-5-phosphonopentanoic acid (50 microM), NO guanylyl cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (10 microM), and the PKG inhibitor KT5823 (1 microM). We further show that LTD induced by low-frequency stimulation in the absence of SNAP also is blocked by KT5823 or Rp-8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphorothioate (10 microM), cyclic guanosine 3',5' monophosphate-dependent protein kinase (PKG) inhibitors with different mechanisms of action. Furthermore SNAP-facilitated LTD was blocked when release from intracellular calcium stores was inhibited by ryanodine (10 microM). Finally, two cell-permeant antagonists of the cyclic ADP-ribose binding site on ryanodine receptors also were able to block the induction of LTD. These results support a cascade for induction of homosynaptic, NO-dependent LTD involving activation of guanylyl cyclase, production of guanosine 3',5' cyclic monophosphate and subsequent PKG activation. This process has an additional requirement for release of Ca2+ from ryanodine-sensitive stores, perhaps dependent on the second-messenger cyclic ADP ribose.     

Inspiratory inhibition and rebound activation elicited by intermittent electrical bulbar stimulation in various states of pulmonary afferent vagal excitation

In anesthetized rabbits spirogram and diaphragmatic activity were examined during electrical stimulation of regions of the medulla oblongata. The volleys were triggered by the animal's own respiration. 1. One volley of 120 msec duration at 100 pulses p.s., applied during inspiratory, caused an immediate and transient inhibition of the diaphragmatic activity. After the end of the volley and inspiratory rebound appeared: the tidal volume was increased and the inspiration was prolonged by some 150 msec. The respiratory rate decreased. 2. Continuous low or high frequency electrical stimulation of pulmonary stretch afferents caused an inspiratory or an expiratory effect respectively. In both conditions the effects of additional intermittent bulbar stimulation remained essentially unaltered. 3. By means of specially designed spirometer both pulmonary collapse and marked lung distension were produced. At low lung volume collapse afferents were excited. Inspiratory inhibition and rebound activation upon central stimulation persisted throughout the whole range of lung volume investigated; the rebound increase in tidal volume consecutive to the stimulus volley, however, was minimal in extreme lung distension. 4. Results suggest that the stimulation effects were the consequence of manipulation on intrinsic mechansims of the bulbar respiratory centre.     

Bilateral soleus H-reflexes in humans elicited by simultaneous trains of stimuli: symmetry, variability, and covariance

Experiments using electrical and mechanical activation of spinal reflexes have contributed important results toward the understanding of neuronal and synaptic dynamics involved in spinal neural circuits as well as their response to different inputs. In this work, data obtained from the simultaneous stimulation of both legs are analyzed to provide information on the degree of symmetry of the respective spinal reflex circuits and on the characteristics of reflex variability. H-reflexes recorded from relaxed muscles show a frequency-dependent amplitude depression when elicited by a train of stimuli. This effect has been attributed to homosynaptic depression. Soleus H-reflexes were recorded in response to trains of simultaneous stimuli applied to both legs in right-handed subjects that were sitting in a relaxed state. The first objective was to verify the existence of asymmetries in H-reflex parameters obtained from the two legs. We measured the mean, variance, and coefficient of variation of the depressed H-reflex amplitudes and the time constant of decay toward the depressed plateau. The second objective was the analysis of the time correlation of subsequent H-reflex amplitudes in a long train of responses recorded from a given leg. The statistical dependence of H-reflex amplitudes in the long trains recorded from both legs was also investigated. Data obtained from preliminary experiments showed that there was no effect of a given stimulus on the contralateral leg applied simultaneously or 1 s before, therefore validating the simultaneous stimulation paradigm. Paired t-tests indicated that several parameters measured bilaterally from soleus H-reflex trains of right-handed subjects were not statistically different in the overall, although individually there were statistically significant asymmetries, toward either the right or left leg. Sequences of H-reflex amplitudes, as measured by the auto-covariance, were either white or had a memory ranging from 2 up to 50 s. This indicates that the random fluctuations in presynaptic inhibition and/or postsynaptic inputs to motoneurons may have either fast or slow time courses. The average auto-covariance sequences of the right and left legs, computed from all subjects, were practically superposable. The cross-covariance between the bilateral H-reflex amplitudes showed a statistically significant peak at zero lag in some experiments, suggesting a correlation between the synaptic inputs to the Ia-motoneuron systems of the soleus muscles of both legs.     

Activation and inhibition of the lateral hypothalamic neurones elicited by medial forebrain bundle stimulation.

1. Single neurone discharges were recorded from the lateral hypothalamus (LH) at the level of the ventromedial nucleus. Single stimulation of the medial forebrain bundle (MFB) at the level of the mesodiencephalic junction elicited a strong inhibition lasting for 100-300 msec in these neurones. 2. However, by the use of multiple stimulating electrodes, it was possible to find a circumscribed locus in the MFB which gave rise to an initial activation of firing discharges with a latency of 1-15 msec (in different neurones) preceding the period of inhibition. 3. One class of the LH neurones was antidromically activated, and was thought to give off axons into the descending MFB. The other was activated orthodromically through a highly efficient synaptic connexion, and was viewed as 'relay' neurones of the ascending MFB. 4. Although the former could also be activated orthodromically, the efficacy of driving was distinctly lower than that observed in the latter. Presumably they integrate the messages from the visceral centres of the medulla and pons, thus participating in the viscero-motor outflow from the hypothalamus. 5. Intracellularly recorded spike potentials of these neurones deteriorated rapidly leaving only hyperpolarization which corresponded in its time course to the suppression of extracellular spike discharges following MFB stimulation. It was suggested that the inhibition was elicited via recurrent routes localized within the hypothalamus.     

Nerve lesions induced by macrophage activation.

The neuropathies associated with infectious processes, including leprosy, retroviral infections and Chagas' disease, represent the largest group of neuropathies in the world. Segmental demyelination and axonal degeneration of nerve fibres are associated with inflammatory infiltrates which contain a large number of mononuclear phagocytes. In order to learn more about the role played by macrophage activation in the nerve lesions observed in inflammatory neuropathies, we have performed a morphological study of nerves injected with products of activation of macrophages including proteolytic enzymes and cytokines (tumour necrosis factor and alpha beta-interferon). We have also studied the effects on nerve fibres of macrophages activated by ingestion of proteose-peptone, a foreign protein, and in the course of a delayed-type hypersensitivity (DTH) reaction. We have found that proteases and urokinase were potent demyelinating agents and that activated macrophages were also able to induce significant demyelination of neighbouring fibres. In contrast, injection of TNF alpha induced more severe nerve lesions consisting of axonal degeneration of the majority of nerve fibres. We thus conclude that infected macrophages which penetrate the endoneurium and macrophages activated in a DTH reaction can both cause neuropathy.     

Complement activation induced by rabbit rheumatoid factor.

Rabbit rheumatoid factor produced in animals by hyperimmunized with group C streptococcal vaccine activated guinea pig complement. Anti-streptococcal serum was fractionated by Sephacryl S-200 chromatography into excluded (19S) and included (7S) material and examined for hemolytic activity in a sensitive homologous hemolytic assay system. In the presence of complement, both 19S and 7S antistreptococcal serum fractions induced lysis of bovine (ox) erythrocytes coated with mildly reduced and carboxymethylated rabbit anti-erythrocyte immunoglobulin G. That rabbit rheumatoid factor was responsible for the observed hemolytic activity was substantiated by hemolytic inhibition assays. Significant inhibition of hemolysis was effected when antistreptococcal serum fractions were incubated in the presence of human immunoglobulin G, rabbit immunoglobulin G, and Fc, whereas, no inhibition was detected when the same fractions were tested in the presence of rabbit Fab or F(ab')2 fragments. Deaggregation of inhibitor preparations revealed a preferential reactivity of rheumatoid factor for rabbit immunoglobulin G. In addition to the rheumatoid factor-dependent hemolytic activity observed in humoral preparations, immunoglobulin G-specific antibody-forming cells in spleen and peripheral blood lymphocyte isolates were enumerated by plaque-forming cell assay.     

TLR3途径诱导多发性骨髓瘤细胞株增殖抑制和凋亡机制研究

目的 研究聚肌苷酸胞苷酸( polyI:C)激活的TLR3通路对多发性骨髓瘤RPMI8226细胞的增殖抑制和凋亡相关的生物学作用及相关机制.方法 RPMI8226细胞培养于RPMI 1640培养基,以不同浓度的polyI:C与该细胞作用不同的时间.收集细胞后,分别利用CCK-8和流式细胞术分析其增殖抑制和凋亡情况,同时抽提总RNA,相对定量PCR测定TLR3通路相关基因表达.结果 PolyI:C对RPMI8226的增殖抑制效应随着作用剂量的增加和时间的延长而增加,24h:12.30％±2.04％、22.50％±2.20％、37.90％±1.30％;48h:17.80％±1.52％、29.60±0.85％、45.80％±1.68％;72 h:25.10％±1.01％、34.60％±1.27％、60.50％±2.08％,差异有统计学意义(P＜0.05).浓度为50、100、200μg/ml的polyl:C与RPMI8226作用48 h后,细胞凋亡率分别为5.60％±1.06％、8.71％±1.06％、13.93％±1.17％,差异具有统计学意义(P＜0.05),而且随着polyI:C作用浓度增加,RPMI8226细胞中TLR3和TRIF mRNA相对于内参β-actin的表达均显著增高,TLR3:1.41±0.10、2.24±0.16、4.08±0.13;TRIF:1.07±0.16、1.97±0.13、3.56±0.19,各组间差异具有统计学意义(P＜0.05).结论 TLR3途径可以有效地抑制多发性骨髓瘤细胞增殖,并且诱导其凋亡,对多发性骨髓瘤的生物治疗具有潜在应用价值.     

Immunosuppression induced by nitric oxide and its inhibition by interleukin-4.

Mice immunized with attenuated Salmonella typhimurium, strain SL3235, while protected against virulent challenge, are unable to mount in vivo and in vitro antibody responses to non-Salmonella antigens, such as tetanus toxoid and sheep red blood cells, and exhibit profoundly suppressed responses to B and T cell mitogens. Suppression of antibody responses is mediated by macrophage (M phi)-released soluble factors, and is completely reversed by treatment with interleukin (IL)-4. The present report identifies the suppressor factor as nitric oxide (NO), and provides evidence for a mechanism by which IL-4 abrogates suppression. Suppressed antibody responses correlated with high levels of NO secretion by splenocytes of SL3235-immunized mice. NO production was observed only in cultures consisting of the adherent cell fraction of immune splenocytes. Further, immunosuppression was reversed by NG-monomethyl-L-arginine (NMLA), a competitive inhibitor of NO synthesis, and was completely blocked by the addition of excess L-arginine. Treatment with IL-4, or anti-interferon (IFN)-gamma monoclonal antibody (mAb), also abrogated suppression. Optimal reversal of suppression was observed only when NMLA, IL-4, or anti-IFN-gamma mAb, was added at day 0 of the 5-day plaque-forming cell assay. Treatment with either IL-4 or anti-IFN-gamma mAb also lead to a sharp inhibition of NO production by immune spleen cells. Moreover, the addition of IL-4 to splenic adherent M phi inhibited their ability to generate NO. Our data characterize an immunoregulatory pathway, involving IFN-gamma and NO, by which M phi mediate immunosuppression and identify IL-4 as a potent inhibitor of this pathway.     

Caspase-1 inhibition attenuates activation of BV2 microglia induced by LPS-treated RAW264.7 macrophages

Neuroinflammation has been recognized as a factor in the pathogenesis of neurodegenerative diseases. Emerging evidence suggests that peripheral inflammation, besides neuroinflammation, functions as a modulator of disease progression and neuropathology in several neurodegenerative diseases. However, detailed correlations among peripheral inflammation, neuroinflammation and neurodegeneration remain unknown. In the present study, we prepared a peripheral inflammation model with lipopolysaccharides (LPS)-stimulated RAW264.7 macrophages to explore its activation on BV2 microglia. We found that LPS induced the production of IL-1β, IL-6 and TNF-α in the culture medium of RAW264.7 macrophages. We further showed that LPS plus ATP activated inflammasome, evidenced by the upregulation of caspase-1 and IL-1β, which was suppressed by ZYVAD, a caspase-1 inhibitor. Furthermore, the conditioned medium obtained from LPS-treated RAW264.7 macrophages activated BV2 microglia, stimulating the release of IL-1β, IL-6 and TNF-α from BV2 cells. ZYVAD pretreatment markedly suppressed BV2 microglia activation induced by RAW264.7 cells conditioned medium. Taken together, our study indicates that macrophage-mediated peripheral inflammation subsequently evokes neuroinflammation and may aggravate neural damage. Inflammasome and caspase-1 may be potential targets for modulating systemic inflammatory responses in neurodegenerative diseases.     

Antibacterial resistance, macrophage influx, and activation induced by bacterial rRNA with dimethyldioctadecylammonium bromide.

Intraperitoneally injected rRNA from Pseudomonas aeruginosa combined with dimethyldioctadecylammonium bromide (DDA) increased nonspecifically the resistance of mice against an intraperitoneal challenge with extracellular (P. aeruginosa, Escherichia coli) and intracellular (Listeria monocytogenes) bacteria. This study concerns the mechanism underlying the nonspecific resistance. RNA with DDA (RNA-DDA) induced a cell influx and activated peritoneal macrophages (M phi) as judged by the decreased 5'-nucleotidase and alkaline phosphodiesterase activities in M phi lysates, the enhanced O2- release, and the increased antitumor activity in comparison with unstimulated M phi. RNA without DDA did not enhance the resistance and did not influence the peritoneal cell numbers or M phi properties. DDA without RNA enhanced the resistance of mice only slightly; it induced a cell influx, yielding elicited M phi as judged by the decreased 5'-nucleotidase activity and increased alkaline phosphodiesterase activity, the slightly enhanced O2- release, and the absence of increased antitumor activity. Both RNA-DDA and DDA M phi showed an enhanced capacity to ingest and kill L. monocytogenes in vitro, DDA M phi being slightly less effective than RNA-DDA M phi with respect to killing. We conclude that the enhanced killing capacity of M phi for L. monocytogenes is characteristic of both elicited DDA M phi and activated RNA-DDA M phi. The relationship between nonspecific resistance, peritoneal cell numbers, and antibacterial M phi activity is discussed. In addition, it is shown that RNA and DDA retain their activity when they are injected apart, suggesting that they activate M phi by sequential action.     

Chemically induced bidirectional differentiation of embryonal carcinoma cells in vitro.

N,N-dimethylacetamide, hexamethylene bisacetamide, and Polybrene induced rapid and extensive differentiation in vitro in an otherwise slowly differentiating subline of embryonal carcinoma cells. The type of differentiated cell induced was dependent on the spatial organization of the stem cells during drug treatment. In monalayer culture "epithelial" cells were produced exclusively. However, treatment of aggregated suspension cultures yielded predominantly "fibroblast-like" cells. The undifferentiated embryonal carcinoma cells and the two differentiated cell types were morphologically distinct when examined by light microscopy, scanning electron microscopy, and transmission electron microscopy; and they had differences in cell surface antigens. Both differential cell types produced large amounts of fibronectin, whereas the embryonal carcinoma cells produced only minimal amounts. This system provides a convenient way to induce relatively synchronous differentiation of embryonal carcinoma cells into specific differentiated cell types.     

Endothelial cell activation induced by tumor necrosis factor and lymphotoxin.

Alterations in the morphology and histochemistry of vascular endothelial cells (EC) have been repeatedly observed at sites of chronic inflammation and immune reactions. These changes, which are most prominent in the EC postcapillary venules present in areas with large lymphocytic infiltrates, include the acquisition of a columnar or cuboidal morphology, the development of ribonuclease-sensitive metachromasia, and an increase in intracellular organelles. Thus, EC at sites of inflammation appear to be activated and to demonstrate increased metabolic activity. This study reports that both tumor necrosis factor-alpha (TNF) and lymphotoxin (LT) can activate cultured human umbilical vein EC, as measured by: 1) increased adhesiveness for lymphocytes, 2) increased cell metabolism, as measured by RNA and protein synthesis, and 3) increased cell volume. Although gamma interferon (IFN-gamma) and interleukin-1 (IL-1) have been shown previously to stimulate EC adhesiveness for lymphocytes, these two cytokines had only marginal effects on EC RNA and protein synthesis, and both caused a decrease in EC volume. These findings suggest that TNF and LT play a role in the type of activation of EC in vivo that leads to the development of tall endothelium and increased lymphocyte emigration.     

Chemically induced mammary gland adenomyoepitheliomas and myoepithelial carcinomas of mice. Immunohistochemical and ultrastructural features.

Myoepithelial cell tumors of the mammary gland have been observed in several mammalian species and are composed of a single cell type (myoepithelium) or, more often, present as a biphasic process including neoplastic ductal epithelial cells. In dogs, these are common tumors, but in humans they are rare neoplasms of the breast, and little is yet known of their pathogenesis, particularly with respect to myoepithelial origin. The present report describes bicellular mammary gland tumors arising from the duct epithelium that were induced in (C57BL/6NCr X DBA/2NCr)F1 (B6D2F1) mice by four weekly oral applications of 1 mg 7,12-dimethylbenz[a]anthracene (DMBA) starting at 8 weeks of age. Mammary tumors developed 7 to 8 months later in 14 of 57 mice, and most showed great morphologic resemblance to human adenomyoepitheliomas and myoepithelial carcinomas. Ultrastructurally, the induced tumors were composed of cuboidal epithelium with a microvillous border originating from the lining duct epithelium and plump oval or highly elongated cells that were identified as myoepithelial in origin. These spindle cells contained abundant microfilaments in parallel orientation, some with focal densities and intermediate filaments that frequently formed loose bundles or compact tonofibrils. The myoepithelial cells possessed well-developed desmosomes and plasma membrane caveolae and were regularly bordered by single or reduplicated basement membranes. By immunohistochemistry, strong immunoreactivity was observed for actin in the myoepithelial tumor component only, whereas cytokeratin was variably present in both duct epithelium and myoepithelium. Neoplastic myoepithelial cells stained purple with phosphotungstic acid hematoxylin (PTAH) and brilliant red with Masson's trichrome. It is suggested that DMBA-induced mouse mammary gland adenomyoepitheliomas and myoepithelial carcinomas may serve as very useful animal models to study myoepithelial tumorigenesis.     

Natural killer cell activation and inhibition by receptors for MHC class I.

Several recent advances have been made in our understanding of the mechanisms which human natural killer cells recognize MHC class I molecules. Three are of special relevance: the identification of a novel molecule (DAP12) with a key role in the activation pathways; the observation that certain immunoglobulin-like receptors for HLA class I molecules are also utilized by other leucocyte lineages; and the definition of MHC class Ib proteins (i.e. HLA-E and Qa-1b) as specific ligands for the phylogenetically conserved CD94-NKG2 lectin-like receptors.     

LTD4 increases cytosolic free calcium and inositol phosphates in human neutrophils: inhibition by the novel LTD4 receptor antagonist,SR2640, and possible relation to modulation of chemotaxis

LTD₄ increased the level of free intracellular calcium ([Ca⁺⁺]_i) and stimulated the production of inositol phosphates (IP) in human polymorphonuclear neutrophils (PMN). Calcium was predominantly mobilized from intracellular pools. After a single stimulus, the cells were refractory to a second challenge with the same concentration of LTD₄, but the calcium response to LTB₄ was normal. The rise in [Ca⁺⁺]_i as well as the stimulated production of IP was inhibited by the novel LTD₄ antagonist, SR2640. SR2640 also abolished the attenuation by LTD₄ of LTB₄-directed PMN chemotaxis. The results suggest that human PMN contain specific LTD₄ receptor that trigger phosphatidyl inositol hydrolysis by activation of phospholipase C, leading to intracellular calcium mobilization, which may be involved in modulation of chemotaxis.     

Complement activation and attack on autologous cell membranes induced by streptolysin-O.

Streptolysin-O damages mammalian membranes through generation of large transmembrane channels formed by membrane-inserted polymers of the toxin (S. Bhakdi et al., Infect. Immun. 47:52-60, 1985). We here report that the native toxin binds naturally occurring human serum immunoglobulin G antibodies to form immune complexes with potent complement-activating capacity. Nanomolar concentrations of toxin added to antibody-containing serum cause rapid consumption of C4 and C5 hemolytic activity and 30 to 90% C3 conversion within 10 to 60 min at 37 degrees C. After binding to target membranes, streptolysin-O polymers serve as foci for antibody-dependent complement activation, which proceeds to completion with the formation of terminal C5b-9 complexes on the autologous cells. The binding and insertion of a primarily water-soluble bacterial product into a host cell membrane has thus been shown to generate a stable and hyperactive focus for activation of and self-attack by the complement system. We suggest that this process perpetuates local tissue damage, deviates host complement action away from the invading bacteria, and may possibly play a role in the pathogenesis of poststreptococcal disease.     

Magnesium ions and ionic channels: activation, inhibition or block--a hypothesis.

The interactions between magnesium ions and ionic membrane channels are complicated and may be classified in three categories: activation, reduction and inhibition of the ionic fluxes across the channels, corresponding to three mechanisms: open-block-close. The interactions between magnesium ions and various ionic channels are reviewed and the explanations of the three mechanisms are analyzed in term of screening/binding effects on the membrane surface polar head groups.