Bladder and cutaneous sensory neurons of the rat express different functional P2X receptors

The expression and functional responses of P2X receptors in bladder and cutaneous sensory neurons of adult rats and mice have been studied using immunohistochemistry and patch clamp techniques. Cell bodies of bladder pelvic afferents were identified in L6 and S1 dorsal root ganglia (DRG), following Fast Blue injection into the muscle wall of the urinary bladder. Similarly, cutaneous sensory neurons were identified in L3 and L4 DRG, following Fast Blue injection into the saphenous nerve innervating the skin. Bladder sensory neurons contained only weak to moderate P2X(3)-immunoreactivity (IR), in contrast to strong P2X(3)-IR observed in a sub-population of cutaneous afferents. Whole-cell patch-clamp recordings revealed that approximately 90% of bladder afferent neurons responded to alpha beta-methylene ATP (alpha beta meATP) and ATP (30 microM) with persistent currents, which were inhibited by 2',3'-O-trinitrophenyl-ATP (TNP-ATP) (0.3 microM) to 6.4+/-1.9% and 8.0+/-2.6% of control, respectively (n=8). The remaining bladder sensory neurons demonstrated biphasic, transient or no response to P2X agonists. In contrast, only 24% of cutaneous afferent neurons gave persistent currents to alpha beta meATP (30 microM), with 66% of cells giving transient or biphasic currents and the remaining 10% being non-responsive. Our results suggest that, in contrast to DRG neurons in general, bladder sensory neurons projecting via pelvic nerves express predominantly P2X(2/3) heteromeric receptors, which are likely to mediate the important roles of ATP as a signaling molecule of urinary bladder filling and nociception.     

Expression of neuronal nicotinic acetylcholine receptors in rat vagal pulmonary sensory neurons

It is known that cigarette smoke inhalation causes airway irritation and cough, and the effect is caused by both direct and indirect stimulatory effects of nicotine on bronchopulmonary sensory nerves. However, little is known about the expression of nicotinic acetylcholine receptors (nAChRs) in these afferents. In the present study, whole-cell patch-clamp recording and RT-PCR were carried out to examine the expression and function of nAChRs in isolated rat vagal pulmonary sensory neurons that were identified by retrograde labeling with a fluorescent tracer. Patch-clamp recordings demonstrated that application of acetylcholine concentration-dependently evoked an inward current in a subset of pulmonary sensory neurons, which was inhibited by hexamethonium. Application of nicotine or 1,1-dimethyl-4-phenylpiperazinium (DMPP) also activated these neurons, evoking an inward current in voltage-clamp configuration and causing depolarization and action potential in current-clamp recordings. RT-PCR analysis further demonstrated the expression of mRNA encoding for the nAChR subunits alpha4, alpha5, alpha6, alpha7, beta2, beta3 and beta4, but not alpha2 and alpha3 in these neurons.     

Functional excitatory synapses in HEK293 cells expressing neuroligin and glutamate receptors

The discovery that neuroligin is a key protein involved in synapse formation offers the unprecedented opportunity to induce functional synapses between neurons and heterologous cells. We took this opportunity recording for the first-time synaptic currents in human embryonic kidney 293 (HEK293) cells transfected with neuroligin and the N-methyl-d-aspartate or AMPA receptor subunits in a co-culture with rat cerebellar granule cells. These currents were similar to synaptic currents recorded in neurons, and their decay kinetics was determined by the postsynaptic subunit combination. Although neuroligin expression was sufficient to detect functional synapses, cotransfection of HEK293 cells with Postsynaptic density-95/synapse-associated protein-90 (PSD-95) significantly increased current frequency. Our results support the central role of neuroligin in the formation of CNS synapses, validate the proposal that PSD-95 allows synaptic maturation, and provide a unique experimental model to study how molecular components determine functional properties of excitatory synapses.     

人趋化因子受体CCR6在HEK293细胞内的稳定表达及其功能分析

目的:构建稳定表达人趋化因子受体6(CCR6)的HEK293细胞株。方法:将CCR6基因和Gα16质粒共转到HEK293细胞中,并挑取稳定表达CCR6基因的HEK293细胞克隆。采用体外趋化实验、钙流实验、RT-PCR、Western blot及免疫荧光染色法,检测CCR6在HEK293细胞表面的表达。结果:经上述实验证实,CCR6基因和Gα16质粒共转染的HEK293细胞上,可稳定表达CCR6,且具有生物学活性。结论:成功地在HEK293细胞表面稳定表达具有生物学活性的CCR6,为研究CCR6的生物学功能及筛选CCR6的拮抗剂奠定了基础。     

Nerve growth factor induces functional nicotinic acetylcholine receptors on rat sensory neurons in culture

Neonatal sensory neurons from rat nodose ganglia express nicotinic acetylcholine receptors when grown in tissue culture without other cell types. The present study investigates the role of nerve growth factor in inducing these receptors. Nerve growth factor has little effect on the growth and survival of nodose neurons in culture, although most neurons were found by quantitative radioautography to have high-affinity nerve growth factor receptors. Nerve growth factor strongly influenced the expression of nicotinic receptors on these neurons: the proportion of acetylcholine-sensitive neurons was approximately 60% in cultures with nerve growth factor compared with 15% in cultures grown without nerve growth factor. The proportion of acetylcholine-sensitive neurons increased over the first week, plateaued by day 12 and remained high for at least three weeks. In contrast, without NGF, the proportion of acetylcholine-sensitive neurons was low throughout the three-week period. The results indicate that nerve growth factor is an important factor in promoting nicotinic receptors on these neurons in culture.     

Characteristics of rat downregulated in adenoma (rDRA) expressed in HEK 293 cells

Studies with apical membrane vesicles have shown that two distinct and separate anion exchange processes are present in rat distal colon, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS)-sensitive exchange, and DIDS-resistant Cl⁻–OH⁻ exchange. These studies proposed that anion exchanger (AE)-1 isoform encodes the former as both apical membrane DIDS-sensitive exchange, and AE1 specific mRNA are present only in surface cells and are downregulated in Na-depleted rats, whereas downregulated in adenoma (DRA) encodes the latter as both DIDS-resistant Cl⁻–OH⁻ exchange, and DRA-specific proteins are present in apical membranes of both surface and crypt cells and are not altered in Na⁺-depleted rats. Studies were, therefore, initiated to identify the function of rat DRA (rDRA) in vitro. rDRA cDNA isolated from rat distal colon encodes a 757-amino-acid protein which has 96 and 81% homology with mDRA and hDRA, respectively. rDRA-specific mRNA expression was detectable only in specific segments of the digestive tract (duodenum, ileum, cecum, proximal colon, and distal colon) but not in the stomach, jejunum, or in the kidney, brain, heart, and lung. HEK 293 cells stably transfected with rDRA exhibited DIDS-insensitive and intracellular acid pH (pH_i 6.5)-sensitive Cl uptake that: (1) was significantly stimulated by outward Cl⁻, , isobutyrate, and possibly OH⁻ gradients; (2) was saturated as a function of increasing extracellular Cl concentrations with an apparent K _m for Cl of 2.9 ± 0.3 mM; and (3) was inhibited competitively by extracellular oxalate but not by . A high rate of DIDS-insensitive Cl influx at pH 6.5 was also present under physiological Cl⁻ concentration. Our observations that rDRA mediates DIDS-insensitive, acid pH-dependent Cl⁻ uptake are consistent with prior observations that rDRA does not mediate DIDS-sensitive exchange in rat distal colon. We speculate that, in addition to mediating pH-sensitive Cl⁻ uptake, rDRA may function as a modifier of other anion transport proteins.     

Lentiviral Vectors Mediate Long-Term and High Efficiency Transgene Expression in HEK 293T cells

Objectives:Lentiviral vectors have been used successfully to rapidly produce decigram quantities of active recombinant proteins in mammalian cell lines. To optimize the protein production platform, the roles of Ubiquitous Chromatin Opening Element (UCOE), an insulator, and selected promoters were evaluated based on efficiency and stability of foreign gene expression mediated by lentiviral vectors.Methods: Five lentiviral vectors, pFIN-EF1α-GFP-2A-mCherH-WPRE containing EF1α promoter and HS4 insulator, p''HR.cppt.3''1.2kb-UCOE-SFFV-eGFP containing SFFV promoter and UCOE, pTYF-CMV(β-globin intron)-eGFP containing CMV promoter and β-globin intron, pTYF-CMV-eGFP containing CMV promoter, and pTYF-EF1α-eGFP with EF1α promoter were packaged, titered, and then transduced into 293T cells (1000 viral genomes per cell). The transduced cells were passaged once every three days at a ratio of 1:10. Expression level and stability of the foreign gene, green fluorescence protein (GFP), was evaluated using fluorescent microscopy and flow cytometry. Furthermore, we constructed a hepatitis C virus (HCV) E1 recombinant lentiviral vector, pLV-CMV-E1, driven by the CMV promoter. This vector was packaged and transduced into 293T cells, and the recombinant cell lines with stable expression of E1 protein were established by limiting dilution.Results:GFP expression in 293T cells transduced with the five lentiviral vectors peaked between passages 3 and 5 and persisted for more than 5 weeks. The expression was prolonged in the cells transduced with TYF-CMV (β-globin intron)-eGFP or TYF-CMV-eGFP, demonstrating less than a 50% decrease even at 9 weeks post transduction (p>0.05). The TYF-CMV-eGFP-transduced cells began with a higher level of GFP expression than other vectors did. The percentage of GFP positive cells for any of the five lentiviral vectors sustained over time. Moreover, the survival rates of all transfected cells exceeded 80% at both 5 and 9 weeks post transduction. Surprisingly, neither the HS4 insulator nor the UCOE sequence improved the GFP expression level or stability. Clonal cell lines with HCV E1 gene were generated from LV-CMV-E1 vector-infected 293T cells. A representative recombinant cell line maintained stable E1expression for at least 9 weeks without significant difference in morphology compared with untreated 293T cells.Conclusion: The results suggest that all five vectors can stably transduce 293T cells, producing long term transgene expression with different efficiencies. However, neither the insulator nor the UCOE improved the GFP expression. The vectors containing the promoter CMV or CMV (β-globin intron) generated the highest gene expressions, manifesting as more favorable candidates for recombinant protein production in HEK293T cells.     

Expression of L-CCR in HEK 293 cells reveals functional responses to CCL2, CCL5, CCL7, and CCL8

It has become clear in the past years that chemokines and chemokine receptors are pivotal regulators of cellular communication and trafficking. In addition to the approximately 20 chemokine receptors that have been cloned and described, various orphan receptors with a chemokine receptor-like structure are known. We have investigated the orphan mouse chemokine receptor (L-CCR) in HEK 293 cells, a receptor that was originally described in a mouse macrophage cell line. Cells expressing this receptor show pertussis toxin-sensitive chemotaxis and small intracellular calcium transients in response to the chemokines CCL2, CCL7, CCL8, and CCL5. Biotinylated CCL2 binds to L-CCR-expressing cells, and transfection experiments with an L-CCR-green fluorescent protein fusion protein showed L-CCR expression in the membranes of recombinant HEK 293 cells. Although radioligand binding was not detected, it is suggested that L-CCR is a functional chemokine receptor.     

Sirt2基因的克隆及其在HEK293中的表达

目的:克隆大鼠Sirt2 基因, 构建其真核表达载体并在HEK293细胞中表达.方法:利用RT-PCR从大鼠脑组织总RNA中扩增出包含Sirt2编码区的cDNA片段, 产物纯化后T-A克隆连接至pMD20-T载体.以此为模板, 将该基因编码区克隆入真核表达载体pcDNA3.1myc-his(-)中, 转染HEK293细胞检测其表达.结果:测序证实所克隆的Sirt2编码区cDNA正确地插入pcDNA3.1myc-his(-)中, 经免疫荧光检测证实其在HEK293细胞中得到表达.结论:成功克隆了大鼠Sirt2 cDNA, 构建了其真核表达载体, 并在HEK293细胞中得到有效表达, 为进一步研究大鼠Sirt2的功能奠定了基础.     

Salusin-a基因真核表达载体的构建及在HEK293细胞中的表达

目的:研究增强型绿色荧光蛋白真核表达载体Salusin-a(pEG-FP-Salusin-a)的构建及在人胚胎肾细胞系293细胞(HEK293)中的表达。方法:提取人单核细胞系THP-1细胞Salusin-a的mRNA,逆转录多聚酶链反应(RT-PCR)扩增Salusin-a基因,克隆入pEGFP-N3载体,双酶切、菌落PCR及测序鉴定pEGFP-Salusin-a质粒。用脂质体转染pEGFP-Salusin-a入HEK293细胞,分为转染细胞组、质粒对照组和空白对照组,检测分析各组荧光蛋白和Salusin-a基因的表达。结果:成功构建pEGFP-Salusin-a质粒,并转染HEK293,细胞转染效率86%,转染细胞组和质粒对照组绿色荧光蛋白的表达明显高于空白对照组(P0.05);转染细胞组Salusin-amRNA的表达明显高于质粒对照组和空白对照组(P0.05)。结论:重组pEGFP-Salusin-a质粒能转染HEK293细胞并表达Salusin-a,为Salusin-a基因功能的进一步研究提供了实验基础。     

Single-channel properties of glycine receptors of juvenile rat spinal motoneurones in vitro

An essential step in understanding fast synaptic transmission is to establish the activation mechanism of synaptic receptors. The purpose of this work was to extend our detailed single-channel kinetic characterization of α1β glycine channels from rat recombinant receptors to native channels from juvenile (postnatal day 12–16) rat spinal cord slices. In cell-attached patches from ventral horn neurones, 1 m m glycine elicited clusters of channel openings to a single conductance level (41 ± 1 pS, n = 12). This is similar to that of recombinant heteromers. However, fewer than 1 in 100 cell-attached patches from spinal neurones contained glycine channels. Outside-out patches gave a much higher success rate, but glycine channels recorded in this configuration appeared different, in that clusters opened to three conductance levels (28 ± 2, 38 ± 1 and 46 ± 1 pS, n = 7, one level per cluster, all levels being detected in each patch). Furthermore, open period properties were different for the different conductances. As a consequence of this, the only recordings suitable for kinetic analysis were the cell-attached ones. Low channel density precluded recording at glycine concentrations other than 1 m m , but the 1 m m data allowed us to estimate the fully bound gating constants by global model fitting of the 'flip' mechanism of Burzomato and co-workers. Our results suggest that glycine receptors on ventral horn neurones in the juvenile rat are heteromers and have fast gating, similar to that of recombinant α1β receptors.     

Block in entry of enteric adenovirus type 41 in HEK293 cells

Human species F adenoviruses, HAdV-40 and HAdV-41, display characteristic gut tropism in vivo as well as poor infectivity in cell culture. To address the hypothesis that poor infectivity of HAdV-40/41 reflects a partial block prior to genome delivery, the internalization and trafficking of HAdV-41, HAdV-5 (species C) and HAdV-35 (species B) were compared in 293 (human embryonic kidney) cells, which complement E1B function in HAdV-40/41, and in A549 (lung epithelial) cells. Unlike fluorescently labeled HAdV-5 virions which were transported towards the nucleus and HAdV-35 virions which colocalized with LAMP-1, HAdV-41 virions appeared to be scattered throughout the cytoplasm but did not colocalize with markers of late endosomes/lysosomes (cathepsin B, LAMP-1) or with caveolin 1. Fluorescent dextran was released from vesicles in only 10% of HAd41-infected cells that took up dextran, compared to 70% of HAdV-5-infected cells, suggesting inefficient disruption of endosomes by HAdV-41 or uptake of HAdV-41 virions into a different compartment than HAdV-5 virions. Quantitative transmission electron microscopy, which showed greater binding of HAdV-41 virions to 293 cells than to A549 cells, identified a major block in uptake of HAdV-41 virions from the surface of both cell lines. More than 80% of virions remained on the surface 60 min p.i. and as late as 4h p.i. In contrast to HAdV-5 and HAdV-35 virions, which associated mostly with clathrin-coated pits, HAdV-41 virions associated mostly with caveolar-like invaginations and, to a lesser extent, with larger non-clathrin-coated pits, suggesting internalization by pathways other than clathrin-mediated endocytosis.     

Functional comparison of HCN isoforms expressed in ventricular and HEK 293 cells

Pacemaker current (I(f)) encoded by the HCN gene family contributes importantly to cardiac rhythm. That contribution depends on the biophysical characteristics of I(f), such as voltage dependence, which vary markedly with cardiac region, development and disease. Heterologous expression studies of individual HCN isoforms have failed to account for the diverse functionality of the native current. To investigate the influence of cellular environment on the gating of HCN channels, we compared the functional characteristics of HCN2 and HCN4, the two major ventricular isoforms, when over-expressed in a normal context (neonatal myocytes) and in a heterologous context (HEK 293 cells). Independent of cell type, HCN4 activates substantially slower than HCN2 and with a half-maximum activation voltage approximately equal 10 mV less negative. However, both isoforms activate more positively in myocytes than in HEK 293 cells. The latter result suggests a context dependence (i.e. cell-type specificity) to HCN voltage dependence that exerts a comparable influence on these two isoforms. This is distinct from the inherent difference in the biophysical properties of HCN2 and HCN4.     

Expression of functional GABAA receptors in neuroendocrine gastropancreatic cells

Gastropancreatic neuroendocrine cells synthesize large amounts of -aminobutyric acid (GABA). This amino acid neurotransmitter appears to be stored in and released from, vesicles similar to small synaptic vesicles. So far, the function of GABA in gastropancreatic, neuroendocrine cells has not been clarified. Previous work suggested that only pancreatic, glucagon-producing ₂ cells contain functional GABA_A receptors. Using subunit-specific antibodies in sections of human antral mucosa, a human gastrinoma and rat pancreas, we show that expression of GABA_A receptors is abundant in gastropancreatic, neuroendocrine cells. Using the patch-clamp technique in the wholecell mode we demonstrate that both the rat insulinoma cell line RIN 38 and the amphicrine cell line AR42J express functional GABA_A receptors, which are characterized by a relatively low benzodiazepine and Zn²⁺ sensitivity and by an insensitivity to the inverse benzodiazepine agonist 6,7--methoxy-4-ethyl--carboline-3-carboxylate (DMCM). In contrast to neurons, activation of GABA_A receptors leads to a membrane depolarization. This depolarization presumably activates voltage-gated Ca²⁺ channels, resulting in an increase in cytosolic Ca²⁺ concentration, [Ca²⁺]_i, as shown with the fluorimetric dye fura-2. The combination of GABA release, GABA_A receptor activation and the [Ca²⁺]_i increase could constitute an autocrine mechanism, modulating the release of hormones such as gastrin, insulin and somatostatin.     

Developmental stability of taurine's activation on glycine receptors in cultured neurons of rat auditory cortex

Taurine is an endogenous amino acid that can activate glycine and/or gamma-aminobutyric acid type A (GABA(A)) receptors in the central nervous system. During natural development, taurine's receptor target undergoes a shift from glycine receptors to GABA(A) receptors in cortical neurons. Here, we demonstrate that taurine's receptor target in cortical neurons remains stable during in vitro development. With whole-cell patch-clamp recordings, we found that taurine always activated glycine receptors, rather than GABA(A) receptors, in neurons of rat auditory cortex cultured for 5-22 days. Our results suggest that the functional sensitivity of glycine and GABA(A) receptors to taurine is critically regulated by their developmental environments.     

The inhibitory action of glycine on rat cortical neurons.

The action of glycine (Gly) on rat cortical neurons was studied. Gly, iontophoretically applied, hyperpolarized the membrane potential, blocked the firing rate and decreased the amplitude of EPSP-IPSP sequences. Generally the membrane resistance during the aminoacid ejection decreased. The role of Gly in the rat cortical inhibition is discussed.