脂质体介导hHCN2基因转染HEK293细胞

目的建立一种研究离子通道的有效模型。方法采用Lipofacta mine2000脂质体将人的超极化激活的环核苷酸门控（HCN）基因转染人胚胎肾（HEK）293细胞,利用全细胞膜片钳技术检测克隆人HCN2基因的表达。结果pcDNA3-hHCN2真核表达载体转染HEK293细胞3d后,全细胞膜片钳技术记录到克隆人HCN2基因编码的通道电流。结论全细胞膜片钳技术稳定、可靠,可为开展克隆离子通道结构和功能关系研究提供基础。     

Genotoxicity of mesoporous silica nanoparticles in human embryonic kidney 293 cells

Mesoporous silica nanoparticles (MSNs) have been widely evaluated for their potential use as carriers for cancer diagnosis and therapy. Understanding the toxicity of MSNs is crucial to their biomedical applications. Although several groups have reported the cytotoxicity of MSNs, the genotoxicity (inducing genetic aberrations) of MSNs in normal human cells has not been extensively investigated. Gene amplification and mutation may initiate and promote carcinogenesis, and changes in mRNA expression can affect normal human physical functions. In this study, human embryonic kidney 293 (HEK293) cells were treated overnight with MSNs at a concentration of 120 µg/mL. The cells were assayed with fluorescent in situ hybridization to check for chromosome changes and gene amplification. Mutations in the epidermal growth factor receptor 1 (EGFR1) and KRAS genes were checked with DNA sequencing. The effects of MSNs on mRNA expression were investigated with an Agilent human mRNA microarray. No chromosomal alterations or gene mutations in EGFR or KRAS were observed in the control HEK293 cells or HEK293 cells exposed to MSNs. The microarray analysis showed that MSNs significantly altered gene expression. The expression of 579 genes was upregulated and that of 1263 genes was downregulated in HEK293 cells treated with MSNs compared with the control HEK293 cells. Our findings suggest that exposure to MSNs is genotoxic to normal human cells, leading to changes in the expression of some genes. This genotoxicity may cause cellular dysfunction and certain benign diseases. We have not shown that MSN exposure induces serious genotoxicity involving carcinogenesis. Copyright © 2015 John Wiley & Sons, Ltd.     

Agents that elevate cyclic AMP induce receptor phosphorylation primarily at serine 331 in HEK 293 cells overexpressing human thromboxane receptor alpha

Human embryonic kidney (HEK) 293 cells stably transfected with the His-tagged thromboxane receptor alpha (TPalpha) were used to study the phosphorylation and desensitization of the receptor induced by prostaglandin E1 (PGE1) or forskolin. These agents are known to increase the intracellular level of cyclic AMP (cAMP) and activate cAMP-dependent protein kinase (PKA). Pretreatment of cells with either agent significantly attenuated Ca2+ release induced by the agonist [1S-[1alpha,2alpha(Z),3beta(1E,3S),4alpha]]-7-[3-[3-hydroxy-4-(4-indophenoxy)-1-butenyl]-7-oxabicyclo[2,2,1]hept-2-yl]-5-heptenoic acid (I-BOP). These agents also induced concentration-dependent phosphorylation of TPalpha as demonstrated by increased 32P-labeling of the receptor from cells prelabeled with 32P(i). To facilitate the identification of the intracellular domains involved in phosphorylation, glutathione S-transferase (GST)-intracellular domain fusion proteins were used as substrates for purified PKA. It was found that only the C-terminal tail fusion protein could serve as a substrate for PKA. To identify the specific serine/threonine residues in the C-terminal tail that are involved in phosphorylation, various alanine mutants of these residues were checked for their ability to serve as substrates. Ser-331 was found to be involved in PKA-mediated phosphorylation. The S331A mutant receptor overexpressed in HEK 293 cells was not phosphorylated significantly following stimulation by PGE1 or forskolin, indicating that Ser-331 was the major site of phosphorylation. Furthermore, cells overexpressing the mutant receptor became responsive to I-BOP-induced Ca2+ mobilization even after pretreatment with PGE1 or forskolin. These results indicate that Ser-331 is the primary site responsible for the phosphorylation and desensitization of the human TPalpha induced by agents that activate PKA.     

Differential state-dependent modification of rat Na(v)1.6 sodium channels expressed in human embryonic kidney (HEK293) cells by the pyrethroid insecticides tefluthrin and deltamethrin

We expressed rat Na_v1.6 sodium channels in combination with the rat β1 and β2 auxiliary subunits in human embryonic kidney (HEK293) cells and evaluated the effects of the pyrethroid insecticides tefluthrin and deltamethrin on expressed sodium currents using the whole-cell patch clamp technique. Both pyrethroids produced concentration-dependent, resting modification of Na_v1.6 channels, prolonging the kinetics of channel inactivation and deactivation to produce persistent “late” currents during depolarization and tail currents following repolarization. Both pyrethroids also produced concentration dependent hyperpolarizing shifts in the voltage dependence of channel activation and steady-state inactivation. Maximal shifts in activation, determined from the voltage dependence of the pyrethroid-induced late and tail currents, were ~ 25 mV for tefluthrin and ~ 20 mV for deltamethrin. The highest attainable concentrations of these compounds also caused shifts of ~ 5–10 mV in the voltage dependence of steady-state inactivation. In addition to their effects on the voltage dependence of inactivation, both compounds caused concentration-dependent increases in the fraction of sodium current that was resistant to inactivation following strong depolarizing prepulses. We assessed the use-dependent effects of tefluthrin and deltamethrin on Na_v1.6 channels by determining the effect of trains of 1 to 100 5-ms depolarizing prepulses at frequencies of 20 or 66.7 Hz on the extent of channel modification. Repetitive depolarization at either frequency increased modification by deltamethrin by ~ 2.3-fold but had no effect on modification by tefluthrin. Tefluthrin and deltamethrin were equally potent as modifiers of Na_v1.6 channels in HEK293 cells using the conditions producing maximal modification as the basis for comparison. These findings show that the actions of tefluthrin and deltamethrin of Na_v1.6 channels in HEK293 cells differ from the effects of these compounds on Na_v1.6 channels in Xenopus oocytes and more closely reflect the actions of pyrethroids on channels in their native neuronal environment.     

Ca^2＋ participates in α1B-adrenoceptor-mediated cAMP response in HEK293 cells

AIM: To investigate the alpha1B-adrenoceptor (alpha1B-AR)-mediated cAMP response and underlying mechanisms in HEK293 cells. METHODS: Full-length cDNA encoding alpha1B-AR was transfected into HEK293 cells using the calcium phosphate precipitation method, and alpha1B-AR expression and cAMP accumulation were determined by using the saturation radioligand binding assay and ion-exchange chromatography, respectively. RESULTS: Under agonist stimulation, alpha1B-AR mediated cAMP synthesis in HEK293 cells, and blockade by PLC-PKC or tyrosine kinase did not reduce cAMP accumulation induced by NE. Pretreatment with pertussis toxin (PTX) had little effect on basal cAMP accumulation as well as norepinephrine (NE)-stimulated cAMP accumulation. In addition, pretreatment with cholera toxin (CTX) neither mimicked nor blocked the effect induced by NE. The extracellular Ca2+ chelator egtazic acid (EGTA), nonselective Ca2+ channel blocker CdCl2 and calmodulin (CaM) inhibitor W-7 significantly reduced NE-induced cAMP accumulation from 1.59%+/-0.47% to 1.00%+/-0.31%, 0.78%+/-0.23%, and 0.90%+/-0.40%, respectively. CONCLUSION: By coupling with a PTX-insensitive G protein, alpha1BAR promotes Ca2+ influx via receptor-dependent Ca2+ channels, then Ca2+ is linked to CaM to form a Ca2+-CaM complex, which stimulates adenylyl cyclase (AC), thereby increasing the cAMP production in HEK293 cell lines.     

Deletion of the distal COOH-terminus of the A2B adenosine receptor switches internalization to an arrestin- and clathrin-independent pathway and inhibits recycling

Background and purpose:

We have investigated the effect of deletions of a postsynaptic density, disc large and zo-1 protein (PDZ) motif at the end of the COOH-terminus of the rat A_2B adenosine receptor on intracellular trafficking following long-term exposure to the agonist 5′-(N-ethylcarboxamido)-adenosine.

Experimental approach:

The trafficking of the wild type A_2B adenosine receptor and deletion mutants expressed in Chinese hamster ovary cells was studied using an enzyme-linked immunosorbent assay in combination with immunofluorescence microscopy.

Key results:

The wild type A_2B adenosine receptor and deletion mutants were all extensively internalized following prolonged treatment with NECA. The intracellular compartment through which the Gln³²⁵-stop receptor mutant, which lacks the Type II PDZ motif found in the wild type receptor initially trafficked was not the same as the wild type receptor. Expression of dominant negative mutants of arrestin-2, dynamin or Eps-15 inhibited internalization of wild type and Leu³³⁰-stop receptors, whereas only dominant negative mutant dynamin inhibited agonist-induced internalization of Gln³²⁵-stop, Ser³²⁶-stop and Phe³²⁸-stop receptors. Following internalization, the wild type A_2B adenosine receptor recycled rapidly to the cell surface, whereas the Gln³²⁵-stop receptor did not recycle.

Conclusions and implications:

Deletion of the COOH-terminus of the A_2B adenosine receptor beyond Leu³³⁰ switches internalization from an arrestin- and clathrin-dependent pathway to one that is dynamin dependent but arrestin and clathrin independent. The presence of a Type II PDZ motif appears to be essential for arrestin- and clathrin-dependent internalization, as well as recycling of the A_2B adenosine receptor following prolonged agonist addition.     

5-Hydroxytryptamine 5-HT1B receptors inhibiting cyclic AMP accumulation in rat renal mesangial cells

A clonal cell line derived from rat renal mesangial cells was shown to express endogenous 5-hydroxytryptamine (serotonin, 5-HT) receptors that mediate inhibition of cyclic AMP accumulation. These receptors were characterized as being of the 5-HT_1B receptor subtype. 5-HT₁ receptor agonists inhibited forskolin-stimulated cyclic AMP accumulation in rat renal mesangial cells (60–70% maximal inhibition) with the following rank order of potency (mean pEC₅₀ values±SEM, n 3): ergotamine (9.58±0.51)>RU 24969 (8.67±0.23)5-CT (8.42±0.06)CP 93129 (8.15±0.27)>5-HT (7.75±0.11) > sumatriptan (6.29±0.30) > 8-OH-DPAT (4.32±0.15). 5-HT₂ and 5-HT₄ receptor agonists were without effect. 5-HT-induced inhibition of cyclic AMP accumulation was abolished by a pre-treatment of the cells with pertussis toxin. (-)Propranolol was a partial agonist (27% maximal inhibition, pEC₅₀ 7.19±0.24, n = 3); when used as an antagonist at 1 M, it shifted the concentration-response curve of 5-HT to the right (pKB 7.22±0.35, n = 3). Methiothepin was a competitive antagonist of 5-HT (pA₂ 8.04±0.10, Schild slope 0.87±0.21, n = 3). Rauwolscine (10 M) had no antagonist activity. There was a significant correlation (r = 0.98, P = 0.0001) between the cyclic AMP data obtained in rat mesangial cells and 5-HT_1B binding data reported in rat brain cortex. The same pattern of responses was observed in early passages of primary cultures of rat mesangial cells. This study shows that rat mesangial cells can be used as a convenient source of functional 5-HT_1B receptors. It also constitutes further evidence for the widespread distribution of 5-HT_1B receptors outside the brain.     

A new chemical tool (C0036E08) supports the role of adenosine A(2B) receptors in mediating human mast cell activation

Asthma is a chronic inflammatory disease of the airways that involves many cell types, amongst which mast cells are known to be important. Adenosine, a potent bronchoconstricting agent, exerts its ability to modulate adenosine receptors of mast cells thereby potentiating derived mediator release, histamine being one of the first mediators to be released. The heterogeneity of sources of mast cells and the lack of highly potent ligands selective for the different adenosine receptor subtypes have been important hurdles in this area of research. In the present study we describe compound C0036E08, a novel ligand that has high affinity (pK(i) 8.46) for adenosine A(2B) receptors, being 9 times, 1412 times and 3090 times more selective for A(2B) receptors than for A(1), A(2A) and A(3) receptors, respectively. Compound C0036E08 showed antagonist activity at recombinant and native adenosine receptors, and it was able to fully block NECA-induced histamine release in freshly isolated mast cells from human bronchoalveolar fluid. C0036E08 has been shown to be a valuable tool for the identification of adenosine A(2B) receptors as the adenosine receptors responsible for the NECA-induced response in human mast cells. Considering the increasing interest of A(2B) receptors as a therapeutic target in asthma, this chemical tool might provide a base for the development of new anti-asthmatic drugs.     

Loss of constitutive activity is correlated with increased thermostability of the human adenosine A2A receptor

Background and Purpose

Thermostabilization by mutagenesis is one method which has facilitated the determination of high-resolution structures of the adenosine A_2A receptor (A_2AR). Sets of mutations were identified, which both thermostabilized the receptor and resulted in preferential agonist (Rag23 mutant) or antagonist (Rant5 and Rant21) binding forms as assessed by radioligand binding analysis. While the ligand-binding profiles of these mutants are known, the effects these mutations have on receptor activation and downstream signalling are less well characterized.

Experimental Approach

Here we have investigated the effects of the thermostabilizing mutations on receptor activation using a yeast cell growth assay. The assay employs an engineered Saccharomyces cerevisiae, MMY24, which couples receptor activation to cell growth.

Key Results

Analysis of the receptor activation profile revealed that the wild-type (WT) A_2AR had considerable constitutive activity. In contrast, the Rag23, Rant5 and Rant21 thermostabilized mutants all exhibited no constitutive activity. While the preferentially antagonist-binding mutants Rant5 and Rant21 showed a complete lack of agonist-induced activity, the Rag23 mutant showed high levels of agonist-induced receptor activity. Further analysis using a mutant intermediate between Rag23 and WT indicated that the loss of constitutive activity observed in the agonist responsive mutants was not due to reduced G-protein coupling.

Conclusions and Implications

The loss of constitutive activity may be an important feature of these thermostabilized GPCRs. In addition, the constitutively active and agonist-induced active conformations of the A_2AR are distinct.     

A2B adenosine receptors inhibit superoxide production from mitochondrial complex I in rabbit cardiomyocytes via a mechanism sensitive to Pertussis toxin

BACKGROUND AND PURPOSE

A_2B adenosine receptors protect against ischaemia/reperfusion injury by activating survival kinases including extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K). However, the underlying mechanism(s) and signalling pathway(s) remain undefined.

EXPERIMENTAL APPROACH

HEK 293 cells stably transfected with human A_2B adenosine receptors (HEK-A_2B) and isolated adult rabbit cardiomyocytes were used to assay phosphorylation of ERK by Western blot and cation flux through cAMP-gated channels by patch clamp methods. Generation of reactive oxygen species (ROS) by mitochondria was measured with a fluorescent dye.

KEY RESULTS

In HEK-A_2B cells, the selective A_2B receptor agonist Bay 60-6583 (Bay 60) increased ERK phosphorylation and cAMP levels, detected by current through cAMP-gated ion channels. However, increased cAMP or its downstream target protein kinase A was not involved in ERK phosphorylation. Pertussis toxin (PTX) blocked ERK phosphorylation, suggesting receptor coupling to G_i or G_o proteins. Phosphorylation was also blocked by inhibition of PI3K (with wortmannin) or of ERK kinase (MEK1/2, with PD 98059) but not by inhibition of NO synthase (NOS). In cardiomyocytes, Bay 60 did not affect cAMP levels but did block the increased superoxide generation induced by rotenone, a mitochondrial complex I inhibitor. This effect of Bay 60 was inhibited by PD 98059, wortmannin or PTX. Inhibition of NOS blocked superoxide production because NOS is downstream of ERK.

CONCLUSION AND IMPLICATIONS

Activation of A_2B adenosine receptors reduced superoxide generation from mitochondrial complex I through G_i/o, ERK, PI3K, and NOS, all of which have been implicated in ischaemic preconditioning.     

Up-regulation of A(2A) adenosine receptors by proinflammatory cytokines in rat PC12 cells

The purpose of this study was to examine the regulation of A(2A) adenosine receptor (A(2A) AR) gene expression induced by proinflammatory cytokines in PC12 cells. The A(2A) AR mRNA levels were substantially increased following 3-48 hr PC12 cell treatment with interleukin 1 beta (500 unit/mL) or tumor necrosis factor alpha (1000 unit/mL), as revealed by RT-PCR analysis. In parallel, cell cytokine treatment induced an up-regulation of A(2A) receptor protein. Equilibrium radioligand binding studies on treated-cells showed a significant increase in maximum density of [3H] 2-(carboxyethylphenylethylamino) adenosine-5'-carboxamide binding sites, with no significant changes in the affinity constant value. The increase in A(2A) receptor density was also demonstrated by Western blot analysis. Interleukin 1 beta and tumor necrosis factor alpha effects on A(2A) AR mRNA and protein levels were detectable after 3 hr cytokine treatment and reached a maximum within 24 and 48 hr, respectively. These results demonstrated the existence of heterologous regulation of A(2A) ARs by proinflammatory cytokines. The biological significance of this regulation might be associated with modulating cellular activity in response to tissue damage associated with inflammatory mediator production.     

Treating lung inflammation with agonists of the adenosine A2A receptor: promises, problems and potential solutions

Adenosine A(2A) receptor agonists may be important regulators of inflammation. Such conclusions have come from studies demonstrating that, (i) adenosine A(2A) agonists exhibit anti-inflammatory properties in vitro and in vivo, (ii) selective A(2A) antagonists enhance inflammation in vivo and, (iii) knock outs of this receptor aggravate inflammation in a wide variety of in vivo models. Inflammation is a hallmark of asthma and COPD and adenosine has long been suggested to be involved in disease pathology. Two recent publications, however, suggested that an inhaled adenosine A(2A) receptor agonist (GW328267X) did not affect either the early and late asthmatic response or symptoms associated with allergic rhinitis suggesting that the rationale for treating inflammation with an adenosine A(2A) receptor agonist may be incorrect. A barrier to fully investigating the role of adenosine A(2A) receptor agonists as anti-inflammatory agents in the lung is the side effect profile due to systemic exposure, even with inhalation. Unless strategies can be evolved to limit the systemic exposure of inhaled adenosine A(2A) receptor agonists, the promise of treating lung inflammation with such agents may never be fully explored. Using strategies similar to that devised to improve the therapeutic index of inhaled corticosteroids, UK371,104 was identified as a selective agonist of the adenosine A(2A) receptor that has a lung focus of pharmacological activity following delivery to the lung in a pre clinical in vivo model of lung function. Lung-focussed agents such as UK371,104 may be suitable for assessing the anti-inflammatory potential of inhaled adenosine A(2A) receptor agonists.     

Arsenic trioxide (As2O3) induced calcium signals and cytotoxicity in two human cell lines: SY-5Y neuroblastoma and 293 embryonic kidney (HEK)

Arsenic trioxide (As(2)O(3)) has anticancer properties; however, its use also leads to neuro-, hepato- or nephro-toxicity, and therefore, it is important to understand the mechanism of As(2)O(3) toxicity. We studied As(2)O(3) influence on intracellular calcium ([Ca(2+)](i)) homeostasis of human neuroblastoma SY-5Y and embryonic kidney cells (HEK 293).We also relate the As(2)O(3) induced [Ca(2+)](i) modifications with cytotoxicity. We used Ca(2+) sensitive dyes (fluo-4 and rhod-2) combined with laser scanning microscopy or fluorescence activated cell sorting to measure Ca(2+) changes during the application of As(2)O(3) and we approach evaluation of cytotoxicity. As(2)O(3) (1 microM) increased [Ca(2+)](i) in SY-5Y and HEK 293 cells. Three forms of [Ca(2+)](i)-elevations were found: (1) steady-state increases, (2) transient [Ca(2+)](i)-elevations and (3) Ca(2+)-spikes. [Ca(2+)](i) modifications were independent from extracellular Ca(2+) but dependent on internal calcium stores. The effect was not reversible. Inositol triphosphate (IP(3)) and ryanodine (Ry) receptors are involved in regulation of signals induced by As(2)O(3). 2-APB and dantrolene significantly reduced the [Ca(2+)](i)-rise (p<0.001, t-test) but did not completely abolish [Ca(2+)](i)-elevation or spiking. This indicates that other Ca(2+) regulating mechanisms are involved. In cytotoxicity tests As(2)O(3) significantly reduced cell viability in both cell types. Staining with Hoechst 33342 showed occurrence of apoptosis and DNA damage. Our data suggest that [Ca(2+)](i) is an important messenger in As(2)O(3) induced cell death.     

Reduced appetite for caffeine in adenosine A(2A) receptor knockout mice

Adenosine A(2A) receptor knockout mice (A(2A)R KO) were compared to wild-type controls (A(2A)R WT) in a caffeine intake paradigm. When mice had ad libitum access to caffeine (0.3 g/l) and water in a two-bottle paradigm for 12 consecutive days, adenosine A(2A)R KO mice drank less caffeinated solution, demonstrating a reduced appetite for caffeine as compared to adenosine A(2A)R WT mice. These data reveal an important role for the adenosine A(2A) receptor in the appetitive properties of caffeine.     

[3H]-SCH 58261 labelling of functional A2A adenosine receptors in human neutrophil membranes

1. The present study describes the direct labelling of A_2A adenosine receptors in human neutrophil membranes with the potent and selective antagonist radioligand, [³H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4 triazolo[1,5-c]pyrimidine, ([³H]-SCH 58261). In addition, both receptor affinity and potency of a number of adenosine receptor agonists and antagonists were determined in binding, adenylyl cyclase and superoxide anion production assays.
2. Saturation experiments revealed a single class of binding sites with K_d and B_max values of 1.34 nM and 75 fmol mg⁻¹ protein, respectively. Adenosine receptor ligands competed for the binding of 1 nM [³H]-SCH 58261 to human neutrophil membranes, with a rank order of potency consistent with that typically found for interactions with the A_2A adenosine receptors. In the adenylyl cyclase and in the superoxide anion production assays the same compounds exhibited a rank order of potency identical to that observed in binding experiments.
3. Thermodynamic data indicated that [³H]-SCH 58261 binding to human neutrophils is entropy and enthalpy-driven. This finding is in agreement with the thermodynamic behaviour of antagonists binding to rat striatal A_2A adenosine receptors.
4. It was concluded that in human neutrophil membranes, [³H]-SCH 58261 directly labels binding sites with pharmacological properties similar to those of A_2A adenosine receptors of other tissues. The receptors labelled by [³H]-SCH 58261 mediated the effects of adenosine and adenosine receptor agonists to stimulate cyclic AMP accumulation and inhibition of superoxide anion production in human neutrophils.

     

Glutamate efflux from human cerebrocortical slices during ischemia: vesicular-like mode of glutamate release and sensitivity to A(2A) adenosine receptor blockade

Glutamate extracellular accumulation is an early event in brain ischemia triggering excitotoxic neuron damage. We have investigated how to control the glutamate efflux from human cerebrocortical slices superfused in conditions simulating an acute ischemic insult (oxygen and glucose deprivation). The efflux of previously accumulated [3H]D-aspartate or endogenous glutamate increased starting 18 min after exposure to ischemia and returned almost to basal values in 6 min reperfusion with standard medium. Superfusion with Ca2+-free, EGTA (0.5 mM)-containing medium or with medium containing tetrodotoxin (TTX; 0.5 microM) inhibited the ischemia (24 min)-evoked [3H]D-aspartate efflux by about 50% and 65%, respectively. The ischemia (24 or 36 min)-evoked efflux of [3H]D-aspartate or endogenous glutamate was reduced at least 40% by the adenosine A(2A) receptor antagonist SCH 58261 (1 microM); the compound was effective when added up to 15 min after exposure to ischemia. No effect of SCH 58261 on the ischemia-evoked [3H]D-aspartate was found in Ca2+-free, EGTA-containing medium. To conclude, a significant component of the ischemia-evoked glutamate efflux in human cerebrocortical slices seems to occur by a vesicular-like mechanism. Endogenously released adenosine is likely to activate A(2A) receptors that enhance vesicular-like glutamate release during ischemia; A(2A) receptor antagonists would deserve consideration for their neuroprotective potential.     

In vitro induction of T cells that are resistant to A2 adenosine receptor-mediated immunosuppression

Background and purpose:

The increased levels of extracellular adenosine in inflamed tissues down-regulate activated immune cells via the A_2A adenosine receptor. This A_2A adenosine receptor-mediated immunosuppression is a disqualifying obstacle in cancer immunotherapy as it protects cancerous tissues from adoptively transferred anti-tumour T cells. The aim of this study was to test whether the negative selection of T cells will produce T cells that are resistant to inhibition by extracellular adenosine.

Experimental approach:

Cytotoxic T lymphocytes (CTL) were developed by mixed lymphocyte culture in the presence or absence of the adenosine receptor agonist 5′-N-ethylcarboxamidoadenosine (NECA). The sensitivity of CTL to adenosine analogues was characterized by cAMP induction, interferon-γ production and cytotoxicity.

Key results:

CTL that could proliferate even in the presence of NECA were less susceptible to inhibition by A_2A adenosine receptor agonists, as shown by a much smaller accumulation of cAMP and less inhibition of interferon-γ production compared with control CTL. The successful protocol to produce CTL that are both resistant to adenosine-mediated immunosuppression and maintain strong cytotoxicity and interferon-γ secretion required NECA to be added only during the expansion stage after the establishment of CTL. In contrast, the priming of resting T cells in the presence of NECA resulted in T cells with impaired effector functions.

Conclusions and implications:

Adenosine-resistant effector T cells were successfully obtained by exposure of activated T cells to NECA. These in vitro studies form the basis for future attempts to produce anti-tumour T cells that are more effective in adoptive immunotherapy.     

A1 and A2 adenosine receptor modulation of contractility in the cauda epididymis of the guinea-pig

1. The effects of adenosine receptor agonists upon phenylephrine-stimulated contractility and [³H]-cyclic adenosine monophosphate ([³H]-cyclic AMP) accumulation in the cauda epididymis of the guinea-pig were investigated. The α₁-adrenoceptor agonist, phenylephrine elicited concentration dependent contractile responses from preparations of epididymis. In the absence or presence of the L-type Ca²⁺ channel blocker, nifedipine (10 μM) the non-selective adenosine receptor agonist, 5′-N-ethylcarboxamido-adenosine (NECA, 1 μM) shifted phenylephrine concentration-response curves to the left (4 and 5 fold respectively). Following the incubation of preparations with pertussis toxin (200 ng ml⁻¹ 24 h) NECA shifted phenylephrine concentration-response curves to the right (5.7±0.9 fold).
2. In the presence of phenylephrine (1 μM), NECA and the A₁ adenosine receptor selective agonists, N⁶-cyclopentyladenosine (CPA) and (2S)-N⁶-[2-endo-norbornyl]adenosine ((S)-ENBA) elicited concentration-responses dependent contractions from preparations of epididymis (pEC₅₀ values 8.18±0.19, 7.79±0.29 and 8.15±0.43 respectively). The A₃ adenosine receptor agonists N⁶-iodobenzyl-5′-N-methyl-carboxamido adenosine (IBMECA) and N⁶-2-(4-aminophenyl) ethyladenosine (APNEA) mimicked this effect (but only at concentrations greater than 10 μM). In the presence of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 30 nM) CPA concentration-response curves were shifted, in parallel to the right (apparent pK_B 8.75±0.88) and the maximal response to NECA was reduced.
3. In the presence of DPCPX (100 nM) the adenosine agonist NECA and the A_2A adenosine receptor selective agonist, CGS 21680 (2-p-(2-carboxyethyl)-phenethylamino-N-ethylcarboxamido adenosine), but not CPA, inhibited phenylephrine (20 μM) stimulated contractions (pIC₅₀ 7.15±0.48). This effect of NECA was blocked by xanthine amine congener (XAC, 1 μM) and the A_2A adenosine receptor-selective antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385; 30 nM).
4. (S)-ENBA (in the absence and presence of ZM 241385, 100 nM), but not NECA or CPA inhibited the forskolin (30 μM)-stimulated accumulation of [³H]-cyclic AMP in preparations of the epididymis of the guinea-pig (by 17±6% of control). In the presence of DPCPX (100 nM) NECA and CGS 21680, but not (S)-ENBA, increased the accumulation of [³H]-cyclic AMP in preparations of epididymis (pEC₅₀ values 5.35±0.35 and 6.42±0.40 respectively), the NECA-induced elevation of [³H]-cyclic AMP was antagonised by XAC (apparent pK_B 6.88±0.88) and also by the A_2A adenosine receptor antagonist, ZM 241385 (apparent pK_B 8.60± 0.76).
5. These studies are consistent with the action of stable adenosine analogues at post-junctional A₁ and A₂ adenosine receptors in the epididymis of the guinea-pig. A₁ Adenosine receptors potentiate α₁-adrenoceptor contractility, an effect blocked by pertussis toxin, but which may not be dependent upon an inhibition of adenylyl cyclase. The epididymis of the guinea-pig also contains A₂ adenosine receptors, possibly of the A_2A subtype, which both inhibit contractility and also stimulate adenylyl cyclase.

     

Facilitation of noradrenaline release by adenosine A(2A) receptors in the epididymal portion and adenosine A(2B) receptors in the prostatic portion of the rat vas deferens

The adenosine-receptor modulation of noradrenaline release was compared in prostatic and epididymal portions of rat vas deferens. In both portions, tritium overflow elicited by electrical stimulation (100 pulses/8 Hz) was reduced by the adenosine A(1) receptor agonist, N(6)-cyclopentyladenosine, and enhanced by the nonselective receptor agonist, 5'-N-ethylcarboxamidoadenosine, in the presence of the adenosine A(1) receptor antagonist, 1,3-dipropyl-8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 20 and 100 nM). The adenosine A(2A) receptor agonist, 2-p-(2-carboxyethyl)phenethyl-amino-5'-N-ethylcarboxamidoadenosine, increased tritium overflow, but only in the epididymal portion. The enhancement caused by NECA was prevented by the adenosine A(2A) receptor antagonist, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385; 20 nM), in the epididymal and by the adenosine A(2B) receptor antagonist, alloxazine (1 microM), in the prostatic portion. Inhibition of adenosine uptake enhanced tritium overflow in both portions, an effect blocked by ZM 241385 in the epididymal and by alloxazine in the prostatic portion. The results indicate that adenosine exerts an adenosine A(1) receptor-mediated inhibition, in both portions, and facilitation mediated by adenosine A(2A) receptors in the epididymal and by A(2B) receptors in the prostatic portion.