Esenbeckia leiocarpa Engl. inhibits inflammation in a carrageenan-induced murine model of pleurisy

Objectives The aim of this study was to investigate the anti‐inflammatory effects of the crude hydroalcoholic extract (CHE) isolated from Esenbeckia leiocarpa Engl., and fractions and subfractions derived from it. Methods Dried E. leiocarpa Engl. bark was macerated and extracted with ethanol to obtain the CHE. The n‐hexane, ethyl acetate, aqueous and alkaloid fractions, as well as two alkaloid subfractions (polar and nonpolar) were obtained from the CHE. A preliminary analysis using thin‐layer chromatography was performed. Capillary electrophoresis, physical characteristics and spectral data produced by IR analysis and nuclear magnetic resonance (¹H and ¹³C NMR), and mass spectrometry analysis were used to identify and elucidate the structure of the major compounds. Swiss mice were used in a carrageenan‐induced pleurisy model. Pro‐inflammatory parameters (leukocyte and exudate concentrations, myeloperoxidase and adenosine‐deaminase activity, and nitrate/nitrite, interleukin 1β and tumour necrosis factor α levels) were quantified in exudates at 4 h after carrageenan‐induced pleurisy in mice. Key findings The dihydrocorynantheol alkaloid was isolated as the majority compound in the CHE, ethyl acetate and alkaloid fractions, and in the polar and nonpolar alkaloid subfractions. The CHE, fractions and subfractions inhibited the increases in leukocyte and exudate concentrations, myeloperoxidase and adenosine‐deaminase activity, and nitrite/nitrate, interleukin 1β, and tumour necrosis factor α levels (P < 0.05) in the fluid secreted from the pleural cavity of the carrageenan‐treated mice. Conclusions E. leiocarpa Engl. showed significant in vivo anti‐inflammatory action by inhibiting the inflammation caused by carrageenan. This effect may be, in part, due to the dihydrocorynantheol alkaloid, which was identified as the majority compound isolated from E. leiocarpa bark.     

Inflammatory cytokines tumour necrosis factor‐α and interleukin‐8 enhance airway smooth muscle contraction by increasing L‐type Ca2+ channel expression

Inflammation elevates intracellular calcium concentrations ([Ca²⁺]_i) in airway smooth muscle (ASM). The L‐type Ca²⁺ channel (L‐VDCC) plays an important role in regulating Ca²⁺ influx in ASM. However, the role of L‐VDCC in the inflammatory cytokine‐induced pathology of ASM remains unclear. In the present study, we used calcium imaging and isometric tension measurements to assess the role of L‐VDCC in agonist‐induced [Ca²⁺]_i rise and the associated contractions in mouse ASM, and we used immunoblotting to identify L‐VDCC protein expression levels in mouse ASM after exposure to tumour necrosis factor alpha (TNF‐α) or interleukin‐8 (IL‐8). Our results showed that high‐K⁺‐ or carbachol‐induced contractions of mouse ASM were significantly greater after pretreatment with TNF‐α or IL‐8 for 24 hours. Both verapamil and nifedipine, L‐VDCC inhibitors, abolished this increased contraction induced by TNF‐α or IL‐8 pretreatment. Moreover, TNF‐α treatment enhanced carbachol‐induced Ca²⁺ influx in ASM cells, and this effect was abrogated by verapamil. Additionally, immunoblotting results showed that preincubation of mouse ASM with TNF‐α or IL‐8 also enhanced L‐VDCC protein expression. On the basis of these findings, we concluded that proinflammatory cytokines, such as TNF‐α and IL‐8, increase the expression level of L‐VDCC, which in turn contributes to augmented agonist‐induced ASM contractions. This effect of inflammation on L‐VDCC expression in ASM may be associated with airway hyper‐responsiveness and involved in the development of asthma.     

RNAi Silencing of IL‐1β and TNF‐α in the Treatment of Post‐traumatic Arthritis in Rabbits

Post‐traumatic arthritis is a secondary complication to severe joint trauma. With the disease progression, it may eventually lead to osteoarthritis in patients whose age is considerably younger than patients with traditional bone arthritis. The main objective of this study was to explore the feasibility of using lentiviral‐mediated RNA interference silencing of IL‐1β and TNF‐α to treat post‐traumatic arthritis in rabbits. About 48 New Zealand rabbits underwent bilateral knee joint surgery to stimulate traumatic arthritis. They were then randomly divided into four groups of 12 rabbits each. The histopathology of the cartilage was observed, and the changes were assessed by Mankin scoring. ELISA was used to detect the expression of IL‐1β and TNF‐α in the synovial fluid. (i) Compared with the control group, the transfection and co‐transfected groups displayed reduced cartilage damage and speed of degeneration. The co‐transfected group showed the greatest alleviation of symptoms. The Mankin score was statistically different (p < 0.01). (ii) Compared with the control group, the expression of IL‐1β or TNF‐α was reduced in the respective transfection groups (p < 0.01 in both groups) and IL‐1β and TNF‐α were reduced in the co‐transfected group (p < 0.01). The co‐transfected group showed the lowest expression of the three experimental groups of both IL‐1β and TNF‐α (p < 0.01). Lentivirus‐mediated RNA interference can knock down the expression of IL‐1β and TNF‐α in joint fluids and, in a synergistic effect when two siRNAs are co‐transfected, ease cartilage degeneration.     

Synthesis of 1,4‐Anthracene‐9,10‐dione Derivatives and Their Regulation of Nitric Oxide,IL‐1β and TNF‐α in Activated RAW264.7 Cells

Mitoxantrone is an anthracenedione antineoplastic and immunosuppressive agent approved for multiple sclerosis treatment. Novel mono‐ and disubstituted anthraquinone derivatives, analogues of mitoxantrone, were synthesized through the addition of lipophilic amino alcohols and evaluated for their effect on IL‐1β, TNF‐α and nitric oxide production by LPS/IFN‐γ‐stimulated RAW264.7 cells. The disubstituted 1,4‐anthracene‐9,10‐dione 10 showed significant inhibition of nitric oxide, TNF‐α and IL‐1β production at the concentration of 5 μg/mL, with a much lower cytotoxicity than mitoxantrone. The monosubstituted 3 , 4 , 11, 12 and 13 also displayed a moderate to good inhibitory capacity on IL‐1β production. However, the methylated compounds 11, 12 and 13 failed to inhibit the TNF‐α production, and compound 13 was the only one to decrease the production of nitric oxide. None of these derivatives was toxic at the tested concentrations. Compounds 10 and 13 had better inhibitory capacity of the inflammatory mediators analyzed, with reliable viability of the cells.     

Apoptosis,Dysbiosis and Expression of Inflammatory Cytokines are Sequential Events in the Development of 5‐Fluorouracil‐Induced Intestinal Mucositis in Mice

The chemotherapeutic agent 5‐fluorouracil (5‐FU) causes intestinal mucositis with severe diarrhoea, but the pathogenesis is not fully understood. In this study, we investigated the pathogenic effects of 5‐FU in mice, focusing on apoptosis, enterobacteria and inflammatory cytokines. Repeated administration of 5‐FU caused severe intestinal mucositis on day 6, accompanied by diarrhoea and body‐weight loss. TNF‐α expression increased 1 day after exposure to the drug, and spiked a second time on day 4, at which point myeloperoxidase activity and IL‐1β expression also increased. Apoptotic cells were observed in intestinal crypts only on day 1. 5‐FU also induced dysbiosis, notably decreasing the abundance of intestinal Firmicutes while increasing the abundance of Bacteroidetes and Verrucomicrobia. Twice‐daily co‐administration of oral antibiotics significantly reduced the severity of intestinal mucositis and dysbiosis, and blocked the increase in myeloperoxidase activity and cytokine expression on day 6, without affecting apoptosis and TNF‐α up‐regulation on day 1. In cultured colonic epithelial cells, exposure to 5‐FU also up‐regulated TNF‐α expression. Collectively, the data suggest that crypt apoptosis, dysbiosis and expression of inflammatory cytokines are sequential events in the development of intestinal mucositis after exposure to 5‐FU. In particular, 5‐FU appears to directly induce apoptosis via TNF‐α and to suppress intestinal cell proliferation, thereby resulting in degradation of the epithelial barrier, as well as in secondary inflammation mediated by inflammatory cytokines.     

VCP746, a novel A1 adenosine receptor biased agonist,reduces hypertrophy in a rat neonatal cardiac myocyte model

VCP746 is a novel A₁ adenosine receptor (A₁AR) biased agonist previously shown to be cytoprotective with no effect on heart rate. The aim of this study was to investigate the potential anti‐hypertrophic effect of VCP746 in neonatal rat cardiac myocytes (NCM). NCM hypertrophy was stimulated with interleukin (IL)‐1β (10 ng/mL), tumour necrosis factor (TNF)‐α (10 ng/mL) or Ang II (100 nmol/L) and was assessed by ³H‐leucine incorporation assay. VCP746 significantly inhibited IL‐1β‐, TNF‐α‐ and Ang II‐stimulated NCM hypertrophy as determined by ³H‐leucine incorporation. The anti‐hypertrophic effect of VCP746 was also more potent than that of the prototypical A₁AR agonist, N⁶‐cyclopentyladenosine (CPA). Further investigation with the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) cell viability assay showed that neither CPA nor VCP746 had any effect on cell viability, confirming that the reduction in ³H‐leucine incorporation mediated by CPA and VCP746 was not due to a reduction in cell viability. IL‐1β, TNF‐α and Ang II were also shown to increase the mRNA expression of hypertrophy biomarkers, ANP, β‐MHC and α‐SKA in NCM. Treatment with VCP746 at concentrations as low as 1 nmol/L suppressed mRNA expression of ANP, β‐MHC and α‐SKA stimulated by IL‐1β, TNF‐α or Ang II, demonstrating the broad mechanistic basis of the potent anti‐hypertrophic effect of VCP746. This study has shown that the novel A₁AR agonist, VCP746, is able to attenuate cardiac myocyte hypertrophy. As such, VCP746 is potentially useful as a pharmacological agent in attenuating cardiac remodelling, especially in the post‐myocardial infarction setting, given its previously established cytoprotective properties.     

Bis‐(3‐hydroxyphenyl) diselenide inhibits LPS‐stimulated iNOS and COX‐2 expression in RAW 264.7 macrophage cells through the NF‐kB inactivation

Objectives Previously, we reported that diaryl diselenide compounds have strong inhibitory effects on lipopolysaccharide (LPS)‐induced nitric oxide (NO) production in macrophages. In this study, we investigated the molecular mechanisms underlying NO suppression and prostaglandin E₂ (PGE₂) production by diaryl diselenide compounds, bis‐(2‐hydroxyphenyl) diselenide (DSE‐A), bis‐(3‐hydroxyphenyl) diselenide (DSE‐B), bis‐(4‐hydroxyphenyl) diselenide (DSE‐C), dipyridyl diselenide (DSE‐D) and diphenyl diselenide (DSE‐E). Methods The effect of these compounds on NO suppression and PGE₂ production was investigated in RAW 264.7 macrophages. Key findings Our data indicate that of the above, DSE‐B most potently inhibits NO and PGE₂ production, and that it also significantly reduces the releases of tumour necrosis factor (TNF)‐α, interleukin(IL)‐1β and IL‐6. Consistent with these observations, DSE‐B also reduced the protein levels of inducible NO synthase (iNOS) and cyclooxygenase‐2 (COX‐2), and the mRNA levels of iNOS, COX‐2, TNF‐α, IL‐1β and IL‐6. Furthermore, DSE‐B inhibited LPS‐induced nuclear factor‐κB (NF‐κB) activation, which was associated with the prevention of the inhibitor κB‐α (IκB‐α) degradation and a subsequent reduction in nuclear p65 protein levels. Conclusions Taken together, our data suggest that the anti‐inflammatory properties of DSE‐B are due to reduction in the expression of iNOS, COX‐2, TNF‐α, IL‐1β and IL‐6 through the down‐regulation of NF‐κB binding activity.     

β‐eudesmol suppresses allergic reactions via inhibiting mast cell degranulation

The regulatory effect of β‐eudesmol, which is an active constituent of Pyeongwee‐San (KMP6), is evaluated for allergic reactions induced by mast cell degranulation. Phorbol 12‐myristate 13‐acetate (PMA) plus calcium ionophore A23187‐stimulated human mast cell line, HMC‐1 cells, and compound 48/80‐stimulated rat peritoneal mast cells (RPMCs) are used as the in vitro models; mice models of systemic anaphylaxis, ear swelling, and IgE‐dependent passive cutaneous anaphylaxis (PCA) are used as the in vivo allergic models. The results demonstrate that β‐eudesmol suppressed the histamine and tryptase releases from the PMA plus calcium ionophore A23187‐stimulated HMC‐1 cells. β‐eudesmol inhibits the expression and activity of histidine decarboxylase in the activated HMC‐1 cells. In addition, β‐eudesmol inhibits the levels of histamine and tryptase released from the compound 48/80‐stimulated RPMCs. Furthermore, β‐eudesmol decreases the intracellular calcium level in the activated RPMCs. β‐eudesmol also decreases the compound 48/80‐induced mortality and ear swelling response. β‐eudesmol suppresses the serum levels of histamine, IgE, interleukin (IL)‐1β, IL‐4, IL‐5, IL‐6, IL‐13, and vascular endothelial growth factor (VEGF) under PCA mice as well as PCA reactions. Therefore, the results from this study indicate the potential of β‐eudesmol as an anti‐allergic drug with respect to its pharmacological properties against mast cell‐mediated allergic reactions.     

Aldosterone induces p21‐regulated apoptosis via increased synthesis and secretion of tumour necrosis factor‐α in human proximal tubular cells

相似文献   

β‐Cyclodextrin‐complexed (−)‐linalool produces antinociceptive effect superior to that of (−)‐linalool in experimental pain protocols

Many plants produce (?)‐linalool, a plant‐derived monoterpene alcohol, including members of the Lamiaceae (mints) and Lauraceae family (laurels, cinnamon, rosewood). The anti‐inflammatory and analgesic effects of (?)‐linalool have been widely suggested for various studies. Poor chemical stability and short half‐life restrain the clinical applications of some essential oil and monoterpenes, including (?)‐linalool. However, β‐cyclodextrin (β‐CD) has been used to increase solubility and stability of lipophilic compounds and also to improve the pharmacological effects. In this study, the antinociceptive effect of (?)‐linalool and (?)‐linalool/β‐CD was examined using the acetic acid writhing reflex, formalin and hotplate tests in rodents. (?)‐Linalool and (?)‐linalool/β‐CD demonstrated strong antinociceptive activity in all the chemical‐ and heat‐induced mice models (p < 0.01 or p < 0.001). These findings imply the involvement of both peripheral and central antinociceptive mechanisms. In peritonitis induced by carrageenan, isolated monoterpene or β‐CD complex also reduced total leucocyte migration and TNF‐α levels in peritoneal fluid. The inclusion complexes, (?)‐linalool/β‐CD, revealed that the antinociceptive effect was significantly (p < 0.01) improved when compared with (?)‐linalool alone. Such results were unlikely to be provoked by any motor abnormality. Together, our results suggest that β‐CD might represent an important tool for improvement of analgesic and anti‐inflammatory profiles of (?)‐linalool and other water‐insoluble compounds, such as lipophilic monoterpenes or essential oils.     

Immunological Response to (1,4)‐α‐d‐Glucan in the Lung and Spleen of Endotoxin‐Stimulated Juvenile Rats

Abstract: We investigated the effects of (1,4)‐α‐D‐glucan (α‐DG), a novel immune stimulatory drug from Tinospora cordifolia, on the concentration of pro‐ and anti‐inflammatory cytokines (interleukin [IL]‐1β, IL‐6, tumour necrosis factor‐α [TNF‐α], γ‐interferon [IFN‐γ] and IL‐10) in the lung and spleen of endotoxin‐stimulated juvenile rats. Experimental groups (n = 16/group) included controls with an intraperitoneal injection of saline, endotoxaemic rats with a non‐lethal dose of 10 mg/kg Escherichia coli endotoxin, and endotoxaemic rats treated with two doses of 10 mg/kg α‐DG, intraperitoneally, 2 and 4 hr after endotoxin injection. At 24 hr of treatment, rats were euthanized and lungs and spleen were removed for cytokines determination and lung injury. Endotoxaemia increased IL‐1β concentration by fivefold in both organs, while creating a moderate pulmonary hypercellularity (demonstrated by about 11% increase in the alveolar‐septal thickening and 11% decrease in the alveolar‐interstitial space ratio). In the lung, α‐DG treatment reduced concentrations of IL‐1β by 30% (p > 0.05), IL‐6 by 43% (p < 0.01), IFN‐γ by 46% (p < 0.01) and the anti‐inflammatory cytokine, IL‐10, by 31% (p > 0.05) compared to endotoxaemia. In the spleen, α‐DG treatment decreased the ratio of IL‐1β to IL‐10 by 55% (p < 0.05), demonstrating an anti‐inflammatory trend. These data suggest that α‐DG differentially modulates cytokine response in the lung and spleen and modifies the pro‐ and anti‐inflammatory balance during an early period of endotoxaemia in juvenile rats.     

Cyanidin‐3‐glucoside inhibits inflammatory activities in human fibroblast‐like synoviocytes and in mice with collagen‐induced arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by joint tissue inflammation. Cyanidin‐3‐glucoside (C3G) is a major component in the flavonoid family and has shown anti‐inflammatory, anti‐oxidant and anti‐tumour activity. In this study, we investigated the effects of C3G on lipopolysaccharides (LPS)‐induced inflammation on human rheumatoid fibroblast‐like synoviocytes (FLS) and on collagen‐induced arthritis (CIA) mice model. We treated FLS with C3G followed by LPS induction, the expressions of tumour necrosis factor alpha (TNF‐α), interleukin 1 beta (IL‐1β) and IL‐6 and the activation of nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) and mitogen‐activated protein kinase (MAPK) signalling pathway were analyzed. CIA was induced in mice and the arthritic mice were treated with C3G for 3 weeks. The disease severity was compared between control and C3G treated mice. The serum levels of TNF‐α, IL‐1β and IL‐6 were analyzed by ELISA. C3G inhibited LPS‐induced TNF‐α, IL‐1β and IL‐6 expression in FLS. Moreover, C3G inhibited LPS‐induced p65 production and IκBa, p38, ERK and JNK phosphorylation. Administration of C3G significantly attenuated disease in mice with CIA and decreased the serum level of TNF‐α, IL‐1β and IL‐6. C3G inhibited LPS‐induced inflammation in human FLS by inhibiting activation of NF‐κB and MAPK signalling pathway. C3G exhibited therapeutic effects in mice with CIA.     

(Pro)renin receptor contributes to diabetic nephropathy by enhancing renal inflammation

1. (Pro)renin receptor (PRR) binding to renin or prorenin mediates angiotensin (Ang) II‐dependent and ‐independent effects. Expression of the PRR is increased in kidneys of diabetic rats, but its role in diabetic nephropathy is unknown. In the present study, we investigated the contribution of the PRR to the development of diabetic nephropathy through enhancement of renal production of tumour necrosis factor (TNF)‐α and interleukin (IL)‐1β. 2. Normoglycaemic control and streptozotocin‐diabetic Sprague‐Dawley rats were used in the study. The urine albumin : creatinine ratio (UACR), renal interstitial fluid (RIF) levels of AngII, TNF‐α and IL‐1β and renal expression of TNF‐α and IL‐1β were evaluated in control, untreated diabetic and diabetic rats treated with either a PRR blocker (PRRB; 0.2 mg/kg per day NH3‐RILLKKMPSV‐COOH), the AT₁ receptor antagonist valsartan (2 mg/kg per day) or combined therapy, administered directly into the renal cortical interstitium for 14 days via osmotic minipumps. 3. Compared with values in normoglycaemic control rats, UACR and RIF AngII, TNF‐α and IL‐1β were significantly higher in untreated diabetic rats. Treatment of diabetic rats with the PRRB or valsartan alone and in combination significantly reduced UACR and RIF TNF‐α and IL‐1β levels. Renal expression of TNF‐α and IL‐1β was higher in untreated diabetic rats than in control rats, but was reduced significantly following treatment with PRRB or valsartan alone and in combination. Renal PRR expression was increased in untreated and PRRB‐treated diabetic rats and reduced in rats receiving valsartan alone or combination therapy. The PRRB had no effect on RIF AngII levels, whereas valsartan alone and in combination with the PRRB significantly increased AngII levels. 4. In conclusion, the PRR is involved in the development and progression of kidney disease in diabetes by enhancing renal production of the inflammatory cytokines TNF‐α and IL‐1β, independent of renal AngII effects.     

Size‐dependent superparamagnetic iron oxide nanoparticles dictate interleukin‐1β release from mouse bone marrow‐derived macrophages

Superparamagnetic iron oxide nanoparticles (SPIONs) have been widely investigated for their biomedical applications in magnetic resonance imaging, targeting therapy, cell labeling, etc. It has been well documented that macrophages produce interleukin (IL)‐1β via several signaling pathways, such as inflammasome activation in response to particles including silica, asbestos and urea crystals with lipopolysaccharide priming. However, the size and dose effects of SPIONs on macrophages and the mechanisms remain unclear. In this study, we explored the cytotoxicity and mechanisms of the synthesized SPIONs with different size distributions of 30, 80 and 120 nm, and compared their potential capability in inducing IL‐1β release in mouse bone marrow‐derived macrophages (BMMs). We found that SPIONs induced IL‐1β release in a size‐ and dose‐dependent manner, in which the smallest SPIONs triggered the highest IL‐1β in BMMs. When cellular uptake of SPIONs was inhibited by the actin polymerization inhibitor, cytochalasin D, SPION‐induced IL‐1β release was suppressed in BMMs. Preventing lysosome damage with bafilomycin A1 or CA‐074‐Me also counteracted SPION‐induced IL‐1β release. Moreover, SPION‐activated IL‐1β release was also attenuated by reactive oxygen species scavengers, diphenylene iodonium or N‐acetylcysteine. Our results elucidated the effects of size and dose on the cytotoxicity and mechanisms of IL‐1β release of SPIONs on macrophages, which facilitate the theoretical and experimental application of SPIONs in biotechnology and biomedicine in the future.     

5‐Amino‐3‐methyl‐4‐isoxazolecarboxylic acid hydrazide derivatives with in vitro immunomodulatory activities

Isoxazoles are an important class of compounds of potential therapeutic value. The aim of this study was to determine immunotropic effects of 5‐amino‐3‐methyl‐4‐isoxazolecarboxylic acid hydrazide derivatives on spontaneous and mitogen‐induced lymphocyte proliferation in young and old mice, cytokine production by peritoneal cells as well as possible mechanism of action in a model of Jurkat cells. Three‐month‐old and 13‐month‐old BALB/c mice were used as donors of the cells from a thymus, a spleen, mesenteric lymph nodes, and a peritoneal cavity. Spontaneous and concanavalin A or lipopolysaccharide (LPS)‐induced cell proliferation was measured by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide colorimetric method. IL‐1β and TNF‐α production induced by LPS in macrophage‐enriched peritoneal cell cultures was measured by enzyme‐linked immunoassay. 5‐amino‐3‐methyl‐4‐isoxazolecarboxylic acid hydrazide, 01K (4‐phenyl‐1‐(5‐amino‐3‐methylisoxazole‐4‐carbonyl)‐thiosemicarbazide), and 06K (4‐(4‐chlorophenyl)‐1‐(5‐amino‐3‐methylisoxazole‐4‐carbonyl)‐thiosemicarbazide) exhibited regulatory activity in the proliferation tests. Prevailing stimulatory activity of the hydrazide and inhibitory activity of 01K and 06K was observed. Those effects were connected with different influence of the compounds on signaling proteins expression in Jurkat cells. The regulatory effects of the compounds on IL‐1β production were more profound than those on TNF‐α. Differences in the compound activity in young versus old mice were mainly restricted to 01K. Immunosuppressive isoxazole leflunomide and a stimulatory RM‐11 (1,7‐dimethyl‐8‐oxo‐1,2H‐isoxazole [5,4‐e]triazepine) were applied as reference drugs.     

Activation of presynaptic A1‐receptors by endogenous adenosine inhibits acetylcholine release in the guinea‐pig ileum

1 It is well established that presynaptic adenosine A₁‐receptor activation inhibits acetylcholine (ACh) release in the guinea‐pig ileum. The present study extends this observation and examines a possible role for endogenous adenosine in modulating cholinergic nerve function. 2 The actions of the adenosine uptake blocker, dipyridamole, the adenosine deaminase inhibitor, erythro‐9(2‐hydroxy‐3‐nonyl)adenine (EHNA) and the A₁‐receptor antagonist, 1,3‐dipropyl‐8‐cyclopentylxanthine (DPCPX) were examined on electrically evoked neurogenic, cholinergic twitch contractions of the guinea‐pig ileum. Some additional studies measuring [³H]‐ACh release were also performed. 3 Adenosine and the selective A₁‐receptor agonist, 2‐chloroadenosine (2‐CA), inhibited electrically evoked contractions and, in the case of 2‐CA, [³H]‐ACh release. The actions were antagonized by DPCPX. At low concentrations, dipyridamole and EHNA enhanced the effect of adenosine causing a leftward shift of the concentration–response curve. In contrast, inhibition induced by 2‐CA was unaffected by either dipyridamole or EHNA. 4 When applied alone at higher concentrations, EHNA and dipyridamole produced a concentration‐dependent suppression of cholinergic neurotransmission. In both cases, the effect could be reversed by DPCPX. At the same concentration, DPCPX alone produced a small but consistent increase in twitch height and [³H]‐ACh release. 5 The data confirm the existence of inhibitory presynaptic adenosine A₁‐receptors modulating cholinergic nerve function in the guinea‐pig ileum and suggests that these receptors can be activated by endogenous adenosine released either as adenosine itself or as an ATP metabolite.