Inhibition of lobster olfactory receptor cells by an odor-activated potassium conductance

1. Whole cell current-clamp recordings show that odors not only depolarize but may also hyperpolarize lobster olfactory receptor cells. Odor-evoked hyperpolarizations occurred in 36% of 178 receptor cells examined. Cell-attached recordings of action potentials followed by current-clamp recordings in the same cell indicate that depolarizing and hyperpolarizing responses were associated with increases (excitation) and decreases (inhibition) in action potential frequency, respectively. Since odorants that hyperpolarized one receptor cell depolarized other cells and since individual cells may be both excited and inhibited, the inhibitory and excitatory nature of the response must be conferred by the odorant-receptor and transduction processes expressed by the receptor cell. 2. The input resistance dropped from 1.73 G omega at rest to 1.45 G omega during odor-evoked hyperpolarization, and the membrane time constant correspondingly decreased from 114 to 61 ms. The increased conductance persisted throughout the stimulation period (5 s). 3. Shifting the K+ reversal to a more negative potential by lowering the [K+]o from 14 to 2.8 mM increased the magnitude of hyperpolarization. The hyperpolarization could be reversibly blocked by dendritic treatment with 5-10 mM 4-aminopyridine (4-AP) or 10 mM cesium ion, but not by 10 mM tetraethylammonium (TEA). 4. Substituting 80% of the [Cl-]o with NO3- increased the amplitude of the hyperpolarization. Based on a calculated equilibrium potential of -32 mV for chloride, an increase in chloride conductance in a low [Cl-]o environment should have decreased the magnitude of the response. Presumably the change in [Cl-]o acts through the dendritic steady-state chloride conductance to shift the membrane potential further from the reversal potential for K+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation-dependent interaction of the N-methyl-D-aspartate receptor epsilon 2 subunit with phosphatidylinositol 3-kinase

BACKGROUND: The NMDA receptors (NMDARs) are ion channels through which Ca2+ influx triggers various intracellular responses. Tyrosine phosphorylation of NMDARs regulates NMDA channel activities, which may be important in neuronal plasticity. The biological significance of the tyrosine phosphorylation events, however, differs among NMDAR subunits: tyrosine phosphorylation of NMDARepsilon1 increases NMDA channel activities, but that of NMDARepsilon2 does not. Since signal transductions from various cell surface receptors are mediated by protein-protein interaction through phosphotyrosine and the Src homology 2 (SH2) domain, we examined the possibility that phosphotyrosines in NMDARepsilon2 contribute to the intracellular signalling events. RESULTS: We first show that Fyn is deeply involved in the phosphorylation of NMDARepsilon2 and second that a phosphotyrosine in NMDARepsilon2 interacts with the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3-kinase). Both the level of tyrosine phosphorylation on NMDARepsilon2 and the amounts of the p85 subunit (p85) bound to NMDARepsilon2 are decreased in Fyn-deficient mice. Moreover, we show that ischaemia stimulates the binding of p85 to phosphorylated NMDARepsilon2, suggesting a physiological role of the phosphotyrosine/SH2-based interaction between NMDARepsilon2 and p85 in the brain. CONCLUSIONS: The tyrosine phosphorylation event on NMDARs is important in not only the regulation of its channel activity but also intracellular signalling mediated through the interaction of the NMDAR with SH2 domain-containing molecules.     

磷脂酰肌醇3-激酶在血管新生相关性疾病中的作用

血管新生参与了人体多种生理与病理过程,前者如创伤愈合,后者包括肿瘤、动脉粥样硬化(atherosclerosis,AS)、类风湿性关节炎(rheumatoid arthritis,RA)等多种疾病,血管新生与这些疾病的发生发展密切相关.磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase, PI3K)作为细胞内重要的信号蛋白,可介导细胞迁移、增殖与血管新生等多种生物学行为的信号转导.     

CD28-dependent killing by human YT cells requires phosphatidylinositol 3-kinase activation

CD28/B7 interactions have been demonstrated to provide a co-stimulatory signal for the generation of CD8⁺ cytotoxic T lymphocytes in the absence of CD4⁺ T helper cells. The CD28 signals required for induction of cytotoxicity have yet to be described. To investigate further the biochemical signaling pathways associated with CD28-dependent cytotoxicity, we have studied the human thymic leukemia cell line, YT. YT cells kill B7⁺ targets in a non-major histocompatibility complex (MHC)-restricted, CD28-dependent manner. CD28 ligation on the surface of YT cells caused a rapid increase in the tyrosine phosphorylation of four major cellular substrates with masses estimated to be 110, 95, 85, and 44 kDa. The 110 and 85 kDa substrates were identified as the catalytic and regulatory subunits, respectively, of phosphatidylinositol 3-kinase (PI3-K). Engagement of CD28 caused the rapid receptor association and activation of PI3-K but did not activate phospholipase C_γ. CD28-induced tyrosine phosphorylation and PI3-K activation was independent of p56^lck protein tyrosine kinase (PTK) activity (previously reported to be associated with CD28) and was insensitive to inhibition by the PTK inhibitor herbimycin A. Two structurally and mechanistically dissimilar inhibitors of PI3-K, wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) also failed to block CD28-dependent tyrosine phosphorylation events or the association of PI3-K with the CD28 receptor. However, both drugs inhibited CD28-dependent cytotoxicity and CD28 receptor associated PI3-K activity with IC₅₀ values similar to the reported IC₅₀ values for PI3-K inhibition. Although herbimycin A did not significantly block the observed CD28-dependent tyrosine phosphorylation or PI3-K activation, herbimycin did block CD28-dependent cytotoxicity in a dose-dependent manner. These data support a role for PI3-K activation in the CD28-dependent initiation of cytotoxic effector function and suggest that a herbimycin sensitive step(s) is either CD28-independent, resides within a PI3-K-independent CD28 signaling pathway, or is downstream of CD28-dependent PI3-K activation.     

Whole cell recording from lobster olfactory receptor cells: responses to current and odor stimulation

1. The basic electrical properties of olfactory (antennule) receptor cells were studied in an in situ preparation of the spiny lobster using whole cell patch-clamp recording. 2. The current-voltage relationship of the cells was linear for membrane potentials between -150 and -40 mV and rectified at more positive membrane potentials. The input resistance at rest averaged 508 M omega. The cells displayed two time constants, with mean values of 29.8 and 8.2 ms. 3. Depolarizing current steps elicited fast, overshooting action potentials at a mean threshold of -32 mV from an imposed resting membrane potential of -65 mV. The action potentials were tetrodotoxin (TTX) and tetraethylammonium (TEA) sensitive, suggesting they are typical sodium/potassium action potentials. 4. Odor stimulation evoked slow, dose-dependent, depolarizing receptor potentials up to 50 mV in amplitude. In approximately 30% of cells tested, these led to repetitive spiking when the cells were depolarized beyond -45 to -30 mV. The amplitude of the receptor potential was graded as a linear function of the logarithm of the odor concentration. 5. The amplitude of the receptor potential varied linearly with the membrane potential between -70 and -30 mV. Extrapolated reversal potentials appeared to be normally distributed around a mean value of -3.6 mV. 6. The results collectively indicate that lobster olfactory receptor cells have electrical properties similar to, but not necessarily identical with, those currently envisaged for olfactory receptor cells in other species.     

Functional role of phosphatidylinositol 3-kinase in direct tumor lysis by human natural killer cells

Cytotoxicity is a key function of natural killer (NK) and T cells; yet the molecular mechanism is unclear. We have biological, biochemical and molecular evidence to demonstrate that phosphatidyl-inositol (PI) 3-kinase is critical for direct NK lysis of tumor cells, via control of intracellular granule movement. Tumor cell engagement rapidly activated PI 3-kinase in NK cells within 5 min, as demonstrated by p85 subunit tyrosine phosphorylation and its ability to generate phosphatidylinositol 3-phosphate, PI(3)P, from PI. Wortmannin and LY294002 effectively inhibited NK cells to lyse 51Cr-labeled tumor cells at the same doses that blocked PI-phosphorylating function in tumor-activated NK cells. Immunostaining demonstrated that tumor engagement for only 5 min mobilized perforin and granzyme B from NK cells unidirectionally towards the target, and prior treatment of NK cells with either PI 3-kinase inhibitor effectively stopped this intracellular polarization. Lastly, ectopic expression of dominant-negative p85 or p110 mutant markedly suppressed NK lytic capacity. These results taken together demonstrate that PI 3-kinase may control NK lytic function via granule polarization towards the contacted target cell.     

Type II phosphatidylinositol 4-kinase beta associates with TCR-CD3 zeta chain in Jurkat cells

Phosphatidylinositol lipid signaling cascades are integral part of TCR-CD3 signaling. The mechanisms by which phosphatidylinositol kinases are coupled to TCR-CD3 complex remain elusive. Here we report an association of type II PtdIns 4-kinase with TCR-CD3 zeta chain upon cross-linking. Mapping studies have revealed that the C-terminal ITAM is critical for docking of the enzyme on the zeta chain. The association is shown to be tyrosyl phosphorylation dependent as mutation of Y-151 and Y-142 on the C-terminal ITAM disrupts interaction of the two proteins. Identification of the associated type II PtdIns 4-kinase revealed that the beta isoform of the enzyme interacts with the zeta chain in vivo.     

FcepsilonRI cross-linking activates a type II phosphatidylinositol 4-kinase in RBL 2H3 cells

Crosslinking of FcepsilonRI on rat basophilic leukemia (RBL 2H3) cells leads to an increase in Phosphatidylinositol 4-kinase activity. This increase in Ptdlns 4-kinase activity is strongly correlated with its tyrosyl phosphorylation state. Characterization of the enzyme activity in anti phosphotyrosine immunoprecipitates suggests it as a type II Ptdlns 4-kinase. Membrane cholesterol depletion studies showed a reduction in type II Ptdlns 4-kinase activity suggesting that lipid rafts play an important role in activation of the enzyme. The enzyme activity was inhibited by resveratrol. In situ inhibition of type II Ptdlns 4-kinase activity showed a reduction in beta-hexosaminidase release upon FcepsilonRI cross-linking. These studies suggest that a type II Ptdlns 4-kinase is an integral component of FcepsilonRI mediated signal transduction mechanisms.     

NGF-induced phosphatidylinositol 3-kinase signaling pathway prevents thapsigargin-triggered ER stress-mediated apoptosis in PC12 cells

Tunicamycin, an inhibitor of the glycosylation of newly biosynthesized proteins, induces endoplasmic reticulum (ER) stress and subsequent apoptosis, and caspase family proteases are activated during the process of ER stress-mediated apoptosis. In the present study, we showed that thapsigargin (Th), an inhibitor of the sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase (SERCA), also induced ER stress-mediated apoptosis, and nerve growth factor (NGF) prevented the apoptosis in PC12 cells. We also found that LY 294002, an inhibitor of phosphatidylinositol 3-kinase (PI 3-K), reduced the survival of cells treated with NGF for 24h in the presence of Th. We discovered that the activities of caspase-3, -9 and -12 were increased time-dependently after the treatment with Th, and NGF suppressed the Th-triggered activation of caspase-3, -9 and -12. LY 294002 diminished the effect of NGF on the inactivation of all these caspases. These results indicate that the NGF-induced PI 3-K signaling pathway prevents Th-triggered ER stress-specific apoptosis via inhibition of caspase-mediated apoptotic signal.     

IL-12 and IL-10 production are differentially regulated by phosphatidylinositol 3-kinase in mast cells

The cellular mechanisms that directly regulate the production of pro- and anti-inflammatory cytokines after lipopolysaccharide (LPS) stimulation in mast cells are currently unresolved. The aim of this study was to clarify the role of phosphatidylinositol 3-kinase (PI3K) in the production of IL-12 and IL-10 in mouse bone marrow-derived mast cells (BMMCs), stimulated with Escherichia coli-derived LPS. LPS activates the PI3K signalling pathway; analysis of cytokine production following LPS stimulation of BMMCs revealed that inhibition of the PI3K pathway differentially regulated IL-10 and IL-12 syntheses. IL-12 production was enhanced, whereas IL-10 levels were suppressed. Inhibition of LPS-mediated activation of the PI3K pathway resulted in a pronounced reduction of NF-κB activity that was dependent on IκBα phosphorylation. These findings demonstrate a regulatory function for PI3K in modulating IL-10 and IL-12 production in mast cells and provide insight into how engagement of the PI3K pathway affects the induction of key immunoregulatory cytokines that control both qualitative and quantitative aspects of early inflammation.     

CD28 of T lymphocytes associates with phosphatidylinositol 3-kinase

T lymphocyte activation requires recognition of antigen by theantigen specific TCR as well as second co-stlmulatory signals.This recognition event results in the activation of non-TCRlinked protein tyroslne klnases (PTKs). The mechanism of co-stlmulatlonof T cells is unknown except for the involvement of PTKs. TheT cell surface molecule CD28 Is effective in delivering co-stlmulatorysignals and prevents T cell anergy by inducing T cell proliferationin TCR stimulated T cells, primarily due to an increase in IL-2production. The mechanism by which CD28 mediates this effectis currently unknown. Some conventional receptor molecules possessintrinsic tyroslne kinase and as a consequence of cross-linkingor llgand binding, phosphorylate numerous tyroslnes within theircytoplasmlc tall, leading these tyroslnes to become ‘activated’and bind cytoplasmlc effector molecules possessing Src homology2 domains which specifically recognize phosphorylated tyroslnes.One such cytoplasmlc effector molecule is the phosphattdyllnosltol-3-phosphatekinase (PI3 kinase) which recognizes the motif phosphotyroslne- methlone/vallne - X - methlonlne (X being any amlno acid)within the cytoplasmlc tails of numerous receptor tyroslne klnases.As CD28 contains a copy of the PI3 kinase binding motif withinits cytoplasmlc tail, we investigated CD28 signaling and PI3kinase activation. Here we demonstrate using the Jurkat cellline that CD28 becomes tyroslne phosphorylated following CD28cross-linking and associates with PI3 kinase. Furthermore, asynthetic peptlde representing the YM/VXM motif within the cytoplasmlctall of CD28 also interacts with PI3 kinase only when the tyroslneis phosphorylated. CD28 co-stimulation, therefore, similar tothat of CD19 and ‘co-stlmulatlon’ of B cells travelin part via the activation of the PI3 kinase pathway.     

Calcium sensitivity of a sodium-activated nonselective cation channel in lobster olfactory receptor neurons

We report that a Na+-activated nonselective cation channel described previously in lobster olfactory neurons, in which phosphoinositide signaling mediates olfactory transduction, can also be activated by Ca2+. Ca2+ activates the channel in the presence of Na+, increasing the open probability of the channel with a K1/2 of 490 nM and a Hill coefficient of 1.3. Ca2+ also increases the sensitivity of the channel to Na+. In some cells, the same channel is Ca2+ insensitive in a cell-specific manner. The nonspecific activator of protein phosphatases, protamine, applied to the intracellular face of patches containing the channel irreversibly eliminates the sensitivity to Ca2+. This effect can be blocked by okadaic acid, a nonspecific blocker of protein phosphatases, and restored by the catalytic subunit of protein kinase A in the presence of MgATP. The Ca2+-sensitive form of the channel is predominantly expressed in the transduction zone of the cells in situ. These findings imply that the Ca2+ sensitivity of the channel, and possibly its regulation by phosphorylation, play a role in olfactory transduction and help tie activation of the channel to the canonical phosphoinositide turnover pathway.     

Myotubularin, a phosphatase deficient in myotubular myopathy, acts on phosphatidylinositol 3-kinase and phosphatidylinositol 3-phosphate pathway

Myotubular myopathy (MTM1) is an X-linked disease, characterized by severe neonatal hypotonia and generalized muscle weakness, with pathological features suggesting an impairment in maturation of muscle fibres. The MTM1 gene encodes a protein (myotubularin) with a phosphotyrosine phosphatase consensus. It defines a family of at least nine genes in man, including the antiphosphatase hMTMR5/Sbf1 and hMTMR2, recently found mutated in a recessive form of Charcot-Marie-Tooth disease. Myotubularin shows a dual specificity protein phosphatase activity in vitro. We have performed an in vivo test of tyrosine phosphatase activity in Schizosaccharomyces pombe, indicating that myotubularin does not have a broad specificity tyrosine phosphatase activity. Expression of active human myotubularin inhibited growth of S.pombe and induced a vacuolar phenotype similar to that of mutants of the vacuolar protein sorting (VPS) pathway and notably of mutants of VPS34, a phosphatidylinositol 3-kinase (PI3K). In S.pombe cells deleted for the endogenous MTM homologous gene, expression of human myotubularin decreased the level of phosphatidylinositol 3-phosphate (PI3P). We have created a substrate trap mutant which shows relocalization to plasma membrane projections (spikes) in HeLa cells and was inactive in the S.pombe assay. This mutant, but not the wild-type or a phosphatase site mutant, was able to immunoprecipitate a VPS34 kinase activity. Wild-type myotubularin was also able to directly dephosphorylate PI3P and PI4P in vitro. Myotubularin may thus decrease PI3P levels by down-regulating PI3K activity and by directly degrading PI3P.     

Clinical development of phosphatidylinositol 3-kinase inhibitors for cancer treatment

The phosphatidylinositol 3-kinase (PI3K) pathway is commonly deregulated in cancer. In recent years, the results of the first phase I clinical trials with PI3K inhibitors have become available. In comparison to other targeted agents such v-raf murine sarcoma viral oncogene homolog B1 (BRAF) inhibitors in melanoma or crizotinib in anaplastic lymphoma receptor tyrosine kinase (ALK) translocated tumors, the number of objective responses to PI3K inhibitors is less dramatic. In this review we propose possible strategies to optimize the clinical development of PI3K inhibitors: by exploring the potential role of PI3K isoform-specific inhibitors in improving the therapeutic index, molecular characterization as a basis for patient selection, and the relevance of performing serial tumor biopsies to understand the associated mechanisms of drug resistance. The main focus of this review will be on PI3K isoform-specific inhibitors by describing the functions of different PI3K isoforms, the preclinical activity of selective PI3K isoform-specific inhibitors and the early clinical data of these compounds.     

磷脂酰肌醇3激酶对哮喘大鼠气道上皮细胞诱导型一氧化氮合酶表达的调控作用

目的： 探讨磷脂酰肌醇3激酶（PI3K）抑制剂wortmannin对哮喘大鼠支气管上皮细胞诱导型一氧化氮合酶（iNOS）表达的影响。方法: 24只成年哮喘大鼠随机分成对照组、哮喘组以及PI3K抑制剂wortmannin干预组。对支气管肺泡灌洗液（BALF）细胞总数及嗜酸性粒细胞进行计数,免疫组织化学检测大鼠支气管上皮细胞iNOS蛋白的表达,RT-PCR检测肺组织iNOS mRNA的表达,分光光度计检测肺组织PI3K活性、iNOS活性及NO含量。结果: 哮喘组大鼠BALF细胞总数计数及嗜酸性粒细胞分类均高于对照组;PI3K抑制剂wortmannin干预组BALF嗜酸性粒细胞计数及分类明显低于哮喘组,差异显著。哮喘组肺组织PI3K活性、iNOS活性及NO含量高于对照组,PI3K抑制剂wortmannin干预组肺组织PI3K活性、iNOS活性及NO含量低于哮喘组。哮喘组大鼠支气管上皮细胞iNOS蛋白及肺组织iNOS mRNA表达较对照组明显增强,但PI3K抑制剂wortmannin组iNOS蛋白及mRNA表达均明显弱于哮喘组。结论: PI3K可调节哮喘大鼠气道iNOS表达,影响哮喘气道炎症反应。     

The role of phosphatidylinositol 4-kinase type IIα in degranulation of RBL-2H3 cells

Objective and Design We have studied the role of phosphatidylinositol 4-kinase IIα (PI4KIIα) in activation of rat basophilic leukemia (RBL-2H3) cells. Materials and Methods Antigen-mediated intracellular Ca²⁺ concentration ([Ca²⁺]_i) increase and β-hexosaminidase secretion were measured using RBL-2H3 cells stably expressing PI4KIIα-yellow fluorescent protein (YFP) or its kinase-deficient mutant PI4KIIα (K151A)-YFP. Results Neither PI4KIIα-YFP nor PI4KIIα (K151A)-YFP were distributed on the plasma membranes but on the exocytotic vesicles. The RBL-2H3 cells stably expressing PI4KIIα-YFP showed significantly enhanced β-hexosaminidase secretion but not an increase in [Ca²⁺]_i after antigen stimulation. The cells with PI4KIIα (K151A)-YFP showed no change in the [Ca²⁺]_i increase nor degranulation. The promotion of secretion by PI4KIIα-YFP was not observed using co-stimulation with Ca²⁺ ionophore and the protein kinase C activator, phorbol myristate acetate. Conclusions These results suggest that PI4KIIα plays a role in the exocytotic process downstream of Ca²⁺ signaling in antigen-mediated mast cell activation. Received 29 September 2005; returned for revision 31 October 2005; accepted by A. Falus 23 May 2006     

Hydrogen peroxide-induced activation of SAPK/JNK regulated by phosphatidylinositol 3-kinase in Chinese hamster V79 cells

To clarify activation mechanisms of stress-activated protein kinase/C-Jun N-terminal kinase (SAPK/JNK) during oxidative stress, the roles of phosphatidylinositol 3-kinase (PI 3-kinase), concentration of intracellular calcium ([Ca2+]i), and cyclic AMP-dependent kinase (PKA) in hydrogen peroxide (H2O2)-induced SAPK/JNK activation were examined in Chinese hamster V79 cells. SAPK/JNK was dose-dependently activated after H2O2 treatment (from 10 microM to 1 mM), and a PI 3-kinase inhibitor (wortmaninn), intracellular calcium chelator (BAPTA-AM), and PKA activator (dibutyl cyclic AMP and forskolin) inhibited this activation. An increase in [Ca2+], was observed after treatment with H2O2. Immunoprecipitation revealed that a PI 3-kinase regulatory subunit, p85alpha, was associated with insulin receptor substance 1 (IRS-1) phosphorylated by H2O2 treatment. Furthermore, the formation of this complex of p85alpha and phospho-IRS-1 was abolished by the presence of BAPTA-AM but not forskolin. These results indicated that the PI 3-kinase activated through phosphorylation of IRS-1 upstream of SAPK/JNK after H2O2 treatment of V79 cells and that [Ca2+]i was a regulation factor for phosphorylation of IRS-1.     

PI-3K在低氧大鼠肺动脉平滑肌细胞增殖中的表达

目的:研究低氧肺动脉平滑肌细胞(PASMC)增殖时磷脂酰肌醇3 激酶(PI-3K)的表达。方法:实验分为无血清培养基(SFM)组和低氧48 h(H48 h)组, 应用免疫组织化学法和流式细胞仪检测低氧PASMC。结果:SFM组和H48 h组PASMC细胞浆内均有PI-3K p110阳性表达, H48 h组(0.1891±0.0301)的表达明显高于SFM组(0.1025±0.0164, P＜0.05);PCNA在SFM组的少数PASMC细胞核内有阳性表达, H48h组(24.58±4.07)%的增殖指数(PI)明显高于SFM组(8.76±1.21)%(P＜0.05);流式细胞仪检测发现PASMC的S+G₂／M期细胞所占细胞总数的百分比在H48 h(27.15±5.43)%显著高于SFM组(10.64±1.52)%(P＜0.05)。结论:低氧可显著上调PI3K的表达, 提示PI-3K可能在低氧PASMC增殖中起重要作用。     

Pharmacological properties and functional role of a TRP-related ion channel in lobster olfactory receptor neurons

Odors activate lobster olfactory receptor neurons (ORNs) through phosphoinositide signaling that appears to target a Na(+)-gated nonselective cation channel. The Na(+)-gated channel is a potential member of the growing family of transient receptor potential (TRP) channels. Here, we test the effect of potential antagonists on the channel in cell-free patches from cultured lobster ORNs. We show that the channel is antagonized by H+ and the TRP channel blockers 2-aminoethoxydiphenyl borate, SKF96365, ruthenium red, Al3+, Gd3+, and La3+. We then use this enhanced antagonist profile together with the agonists Na+ and Ca2+ to implicate the channel in signal amplification in the cells.