Inhibitory Effect Of Caffeine Research Articles

The Effects of Caffeine and Alcohol on the Electrically Evoked Responses of L. terrestris

Regulation on animal research, largely focused on vertebrates, has led to greater expenses for researchers and institutions. Invertebrate animals, however, are rarely included in regulatory oversight, making experimentation comparatively more flexible and inexpensive. Yet, invertebrates are largely overlooked in neurophysiological research, even though the simplicity of invertebrate nervous system models has its advantages. While somatosensory, visual, and auditory evoked potential may be used for pharmacological and toxicological assessment of drugs in both animals and humans, only minor research carried out on invertebrates generally due to the disregard upon comparison between physiological phases or chromosomes of both invertebrates and humans. This study aims to analyze the viability of Lumbricus terrestris, or the common earthworm, as a potential model for electrical somatosensory evoked potential propagation and for drug safety pharmacology assessment. Varying intensities of electrical stimulation potential from 45 ~ 137 mV and accumulative substance injections up to 300 ul were to be used to evaluate telemetric-recorded changes in the evoked potential of the earthworm, and to track the extent of its reproducibility. Alcohol and caffeine, as they are two of the most commonly consumed chemicals in beverages among adolescents, and much was already known about their neurological effects, making the earthworm model’s response to foreign substances evaluable. Numerical changes in latent period, peak point, trough point and wave width of the evoked potential elicited by each substance were analyzed using regression analysis. We found that our peak point and wave width evoked potential parameters were highly indicative of the earthworm’s capability as an evoked potential invertebrate model, especially because responses exhibited expected outcomes of the excitable and inhibitory effects of caffeine and alcohol with high reproducibility.

EFFECT OF CAFFEINE ON SELECTED REPRESENTATIVES OF PHOTOTROPHIC MICROORGANISMS

Aquatic ecosystems are becoming increasingly affected by micropollutants. The survival of microbial communities and entire aquatic ecosystems depends directly on their ability to adapt to this type of pollution. One of the most common micropollutants found in water bodies, especially in the vicinity of large cities, is caffeine. This stimulant directly affects living organisms and can cause changes in the species composition of microbial communities. However, the best way to decontaminate caffeine from the environment may be through the use of microorganisms. Therefore, in this study, the effect of caffeine on selected species of cyanobacteria and algae was investigated. The following strains of cyanobacteria: Geitlerinema cf. acuminatum (CCALA 141), Chlorogloeopsis fritschii (CCALA 039), Chlorogloeopsis fritschii (CCALA 1005), and Synechococcus granulates (CCALA 187) and a eukaryotic photosynthetic flagellate Euglena gracilis (strain Z) were selected. Probit analysis determined the minimum inhibitory concentration (MIC) of caffeine for each studied species. Also, the inhibitory effect of caffeine on each tested strain was monitored in vivo. Next, the strains were microscopically observed, and the potential change in their morphology under the caffeine treatment was recorded. The reproduction rate of all species in the study were moderately inhibited by caffeine, but during the cultivation, they were able to grow in comparison with the control sets. The most sensitive species was Geitlerinema cf. acuminatum (CCALA 141). The study also showed a significant effect of caffeine on morphology changes in the strains under investigation. Caffeine at low concentrations also showed a stimulating effect on the growth of the studied species. This may lead to their improved competence potential within microbial communities in the aquatic biotopes.

Caffeine Inhibits Growth of Temozolomide-Treated Glioma via Increasing Autophagy and Apoptosis but Not via Modulating Hypoxia, Angiogenesis, or Endoplasmic Reticulum Stress in Rats

Thirty rats with glioma were divided into control group, temozolomide (TMZ) group (TMZ 30 mg/kg once daily for 5 day), and TMZ plus Caffeine group (TMZ 30 mg/kg once daily for 5 day and caffeine 100 mg/kg once daily for 2 weeks). The relative tumor fold and expression of hypoxia-induced factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), neuropilin-1 (NRP-1), CCAAT/enhancer-binding protein homologous protein (CHOP), LC-3A/B, apoptosis-inducing factor-1 (AIF-1), and cleaved caspase three were compared. The relative tumor fold of TMZ plus Caffeine group was lower significantly than that of TMZ group at day 14. HIF-1α, VEGF, NRP-1, and CHOP expressions were not significantly different in the three groups. The LC-3A/B expression of TMZ plus Caffeine group was higher significantly than that of the control group and TMZ group. The AIF expressions of TMZ group and TMZ plus Caffeine group were higher significantly than that of the control group. The caspase-3 expression of TMZ plus Caffeine group was higher significantly than that of the control group and TMZ group. In conclusions, the inhibitory effect of caffeine on TMZ-treated glioma might be associated with increasing expressions of autophagy- and apoptosis-related genes.

Evaluation of Inhibitory Effects of Caffeine on Human Carcinoma Cells

One of the world's most consumed medications is caffeine which is available in the vast majority of beverages. Previously, some effects of caffeine have been evaluated including its inhibitory effect on cancer cells. But, the influence of caffeine on esophagus carcinoma squamous cells (CSC) and head and neck carcinoma cells still has not well understood. Herein, the relation between different amounts of caffeine with the proliferation rate of human esophagus carcinoma squamous cell line KYSE-30 as well as human head and neck carcinoma cell line HN5 was evaluated. Furthermore, concentrations of caffeine were adjusted and their effect on cells were studied. The inhibitory effects of caffeine on cells were measured using the conventional colorimetric MTT assay after 3 and 7 day of incubation. Our findings are suggested that caffeine has a significant inhibitory effect on both cell lines at the concentrations of 20, 50, and 70 milli-mol (mmol). The result shows that caffeine can prevent the proliferation of carcinoma cells and it is a perfect candidate for therapeutic applications.

Direct inhibitory effect of caffeine on viability, synthesis activity and gene expression in cultures of chondrocytes extracted from the articular cartilage of rats

ABSTRACT The aim of this study was to evaluate the effect of concentrations of caffeine on the viability, synthesis activity and gene expression in cultures of chondrocytes. Extracted articular cartilage from the femurs and tibias of 15 Wistar rats at three days old to isolate chondrocytes. Chondrocytes were cultured in chondrogenic medium (control) or supplemented with caffeine (0.5, 1.0, 2.0mM). Cell viability, alkaline phosphatase activity and collagen synthesis were assessed using colorimetric assays at 7, 14, 21 days. The chondrocyte cultures of all groups grown under coverslips were stained with hematoxylin-eosin to determine the percentage of cells/field and with PAS, safranin O, alcian blue to determine the percentage of matrix chondrogenic/field at 21 days. The expressions of gene transcripts for aggrecan, collagen-II, Sox-9, Runx-2 and alkaline phosphatase were also evaluated by RT-PCR at 21 days. The means were compared using Student-Newman-Keuls. Caffeine significantly reduced the conversion of MTT to formazan, percentage of cells/field, collagen synthesis, alkaline phosphatase activity, synthesis of PAS+, safranin O+ and alcian blue+ chondrogenic matrix, and the expression of aggrecan, Sox-9 and II collagen. It is concluded that caffeine at concentrations of 0.5, 1.0, 2.0mM has a direct inhibitory effect on chondrogenesis in cultures of chondrocytes from rats.

Evaluation of inhibitory effect of caffeine on bone morphogenesis in pregnancy in a model of rat embryos

Evaluation of inhibitory effect of caffeine on bone morphogenesis in pregnancy in a model of rat embryos

Multiple AMPK activators inhibit l-carnitine uptake in C2C12 skeletal muscle myotubes.

Mutations in the gene that encodes the principal l-carnitine transporter, OCTN2, can lead to a reduced intracellular l-carnitine pool and the disease Primary Carnitine Deficiency. l-Carnitine supplementation is used therapeutically to increase intracellular l-carnitine. As AMPK and insulin regulate fat metabolism and substrate uptake, we hypothesized that AMPK-activating compounds and insulin would increase l-carnitine uptake in C2C12 myotubes. The cells express all three OCTN transporters at the mRNA level, and immunohistochemistry confirmed expression at the protein level. Contrary to our hypothesis, despite significant activation of PKB and 2DG uptake, insulin did not increase l-carnitine uptake at 100 nM. However, l-carnitine uptake was modestly increased at a dose of 150 nM insulin. A range of AMPK activators that increase intracellular calcium content [caffeine (10 mM, 5 mM, 1 mM, 0.5 mM), A23187 (10 μM)], inhibit mitochondrial function [sodium azide (75 μM), rotenone (1 μM), berberine (100 μM), DNP (500 μM)], or directly activate AMPK [AICAR (250 μM)] were assessed for their ability to regulate l-carnitine uptake. All compounds tested significantly inhibited l-carnitine uptake. Inhibition by caffeine was not dantrolene (10 μM) sensitive despite dantrolene inhibiting caffeine-mediated calcium release. Saturation curve analysis suggested that caffeine did not competitively inhibit l-carnitine transport. To assess the potential role of AMPK in this process, we assessed the ability of the AMPK inhibitor Compound C (10 μM) to rescue the effect of caffeine. Compound C offered a partial rescue of l-carnitine uptake with 0.5 mM caffeine, suggesting that AMPK may play a role in the inhibitory effects of caffeine. However, caffeine likely inhibits l-carnitine uptake by alternative mechanisms independently of calcium release. PKA activation or direct interference with transporter function may play a role.

Linoleic Acid Blocks the Inhibitory Effects of Caffeine on Tumour Promotion by 12-O-Tetradecanoylphorbol-13-Acetate in Two-Stage Carcinogenesis in Mouse Skin

The present study assessed the in vivo effects of fatty acids on inflammation and carcinogenesis in mice. Fifteen fatty acids were examined for their effects on the inhibitory effect of caffeine on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear oedema in mice. Furthermore, linoleic acid was studied for its effects on the inhibitory effect of caffeine on carcinogenesis in mouse skin initiated with 7,12-dimethylbenz[a]anthracene (DMBA) and promoted by TPA. Among fatty acids, linoleic, γ-linolenic and oleic acids most strongly blocked the inhibitory effect of caffeine on TPA-induced ear oedema. Furthermore, linoleic acid also markedly blocked the inhibitory effect of caffeine on the tumour-promoting activity of TPA. This is the first report to suggest that fatty acids, such as linoleic, γ-linolenic and oleic acids, block the anti-inflammatory activity of caffeine on TPA-induced inflammation in mice. Linoleic acid blocked the inhibitory effect of caffeine on tumour promotion by TPA in the two-stage mouse skin carcinogenesis model. These results suggest that intake of linoleic acid requires attention.

Downregulation of mPGES-1 ExpressionviaEGR1 Plays an Important Role in Inhibition of Caffeine on PGE2Synthesis of HBx(+) Hepatocytes

We investigated the mechanism of caffeine in influencing HBx(+) hepatocytes to synthesize PGE2. The inhibitory effect of caffeine on hepatocyte proliferation increased with increasing caffeine concentrations (200–800 μM) and treatment times (1–7 days), which was first observed at the second test time point (caffeine treatment for 4 days). The inhibition of caffeine on the growth of HL7702-HBx and HepG2-HBx cells was most obvious at 800 μM caffeine and at caffeine treatment for 7 days. The PGE2 secretion and the expression of mPGES-1 and EGR1 were downregulated, whereas PPARγ expression was upregulated. The mPGES-1 promoter activity of HBx(+) hepatocytes decreased more significantly than that of HBx(−) hepatocytes. Moreover, the expression of EGR1 and PPARγ changed more significantly in HBx(+) hepatocytes cultured for 12 to 24 hours in the presence of 5 mM caffeine. This limited success may be attributed to caffeine releasing the binding of HBx and PPARγ and furthermore affecting the mPGES-1 expression by EGR1 in HBx(+) hepatocytes. The results indicate that caffeine could effectively reduce PGE2 synthesis in HBx(+) hepatocytes by specifically blocking the PPARγ-EGR1-mPGES-1 pathway, thereby providing a new evidence of molecular biology for the hypothesis that drinking coffee is beneficial to HBV-infected patients.

Caffeine Inhibits the Activation of Hepatic Stellate Cells Induced by Acetaldehyde via Adenosine A2A Receptor Mediated by the cAMP/PKA/SRC/ERK1/2/P38 MAPK Signal Pathway

Hepatic stellate cell (HSC) activation is an essential event during alcoholic liver fibrosis. Evidence suggests that adenosine aggravates liver fibrosis via the adenosine A2A receptor (A2AR). Caffeine, which is being widely consumed during daily life, inhibits the action of adenosine. In this study, we attempted to validate the hypothesis that caffeine influences acetaldehyde-induced HSC activation by acting on A2AR. Acetaldehyde at 50, 100, 200, and 400 μM significantly increased HSC-T6 cells proliferation, and cell proliferation reached a maximum at 48 h after exposure to 200 μM acetaldehyde. Caffeine and the A2AR antagonist ZM241385 decreased the cell viability and inhibited the expression of procollagen type I and type III in acetaldehyde-induced HSC-T6 cells. In addition, the inhibitory effect of caffeine on the expression of procollagen type I was regulated by A2AR-mediated signal pathway involving cAMP, PKA, SRC, and ERK1/2. Interestingly, caffeine’s inhibitory effect on the expression of procollagen type III may depend upon the A2AR-mediated P38 MAPK-dependent pathway. Conclusions: Caffeine significantly inhibited acetaldehyde-induced HSC-T6 cells activation by distinct A2AR mediated signal pathway via inhibition of cAMP-PKA-SRC-ERK1/2 for procollagen type I and via P38 MAPK for procollagen type III.

Behavior and changes in cell morphology of Escherichia coli O157:H7 in liquid medium and skim milk in the presence of caffeine

The objective of this study was to investigate the antibacterial effect of caffeine on the behavior and changes in cell morphology of E. coli O157:H7 in a liquid medium and in skim milk. The inhibitory effect of caffeine at different concentrations was determined by inoculating E. coli O157:H7 in laboratory medium and skim milk samples. Samples were incubated at 37ºC for 48 h, and E. coli O157:H7 population was enumerated. Our results showed that caffeine significantly (P < 0.05) inhibited the growth of E. coli O157:H7 in laboratory medium and milk samples. A greater than 3.0 log CFU ml−1 inhibition was observed in milk containing 5.0 g/L caffeine within 12 h of incubation. Moreover, using flow cytometry, marked changes in the morphology of E. coli O157:H7 were also observed. Caffeine has potential as an antimicrobial agent and could be used as an effective natural additive to improve the safety of food products.

AMPK activity is regulated by calcium-mediated protein phosphatase 2A activity

AMP-activated protein kinase (AMPK) is activated by upstream kinases and negatively regulated by protein phosphatases. Intracellular calcium mediates protein phosphatase 2A (PP2A), which is in a heterotrimeric complex with the PR72 subunit. The PR72 subunit contains two calcium-binding sites formed by EF hands. Our previous study has shown that chronic calcium exposure decreases AMPK activity. To define the specific molecular mechanism whereby calcium can deactivate AMPK, activities of AMPK and PP2A were analyzed in C2C12 muscle cell cultures and skeletal muscle tissues from mutant pigs possessing the AMPKγ3-mutation or the ryanodine receptor (RyR1) calcium gating mutation, or both. C2C12 myotubes treated with calcium releasing agent (caffeine) for 10h decreased (P<0.05) AICAR-induced AMPK activity to control levels and this negative effect was eliminated by ryanodine receptor stabilizer, dantrolene. Interestingly, muscle from pigs with the RyR1 mutation and C2C12 cells administered with 10h caffeine showed higher (P<0.05) PP2A activity compared to controls. More importantly, the inhibitory effect of caffeine on AMPK activity was attenuated by the PP2A inhibitor, calyculin A or siRNA induced knockdown of PP2A. These data show the inhibitory effect of chronic calcium on AMPK activity is exerted through the activation of PP2A.

Caffeine-induced endothelial cell death and the inhibition of angiogenesis

Numerous studies have shown that adenosine or adenosine agonists can stimulate angiogenesis. However, the effect of caffeine (a known adenosine receptor antagonist) on angiogenesis has not been previously studied. Accordingly, this study was undertaken to examine the effect of caffeine on angiogenesis and to clarify the mechanism involved. Chick chorioallantoic membrane assays were used to investigate the effect of caffeine on angiogenesis and proliferation assays using human umbilical vein endothelial cells (HUVECs), were used to study its effects on specific aspects of angiogenesis. The expressions of caspase-3 and Bcl-2 were examined by western blotting, immunofluorescence staining was used to identify HUVEC morphological changes, and fluorescence activated cell sorting (FACS) and DAPI staining were used to detect HUVEC apoptosis. Caffeine was found to inhibit blood vessel formation dose-dependently and to inhibit the proliferation of HUVECs time- and dose-dependently. FACS analysis and DAPI staining showed that inhibitory effect of caffeine on HUVEC proliferation was the result of apoptosis and the up-regulation of thrombospondin-1 (TSP-1). Furthermore, TSP-1 levels were down-regulated by NECA but were unaffected by CGS21680, indicating that caffeine regulated TSP-1 expression via adenosine A2B receptor. In addition, caffeine up-regulated caspase-3 and down-regulated Bcl-2 at the protein level. These results suggest that the inhibitory effect of caffeine on angiogenesis is associated, at least in part, with its induction of endothelial cell apoptosis, probably mediated by a caspase-3 dependent mechanism.

Adsorption behavior of caffeine as a green corrosion inhibitor for copper

Electrochemical and impedance experiments were carried out to evaluate the corrosion behavior of copper in aerated 0.1molL−1 H2SO4 solutions in the presence of three xanthine derivatives with similar chemical structures. The corrosion rate of copper was found to increase in the presence of theophylline and theobromine and decrease in the presence of caffeine. The adsorption and inhibitory effect of caffeine on copper surfaces in aerated 0.1molL−1 H2SO4 solutions were then investigated in detail by potentiodynamic polarization, electrochemical impedance spectroscopy (EIS), contact angle measurements, scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS) and fluorescence experiments. The data obtained indicate that caffeine behaves as a cathodic-type inhibitor adsorbing onto the copper surface according to the Temkin isotherm, with the negative ∆G°ads value of −31.1kJmol−1 signifying a spontaneous adsorption process. The corrosion inhibition efficiency increased with caffeine concentration in the range of 1.0–10.0mmolL−1. Furthermore, the EIS results obtained at the open-circuit potential and surface analysis (SEM, EDS and fluorescence) clearly demonstrated the adsorption of the organic compound onto the copper electrode. The contact angle measurements revealed the formation of a hydrophobic protective film. This film covers up to 72% of the total active surface, acts as a protective barrier and prevents interaction between the metal, water and oxygen molecules.

Caffeine Promotes Ultraviolet B-induced Apoptosis in Human Keratinocytes without Complete DNA Repair

In response to ultraviolet B damage, keratinocytes undergo apoptosis to eliminate damaged cells, thereby preventing tumorigenic transformation. Caffeine, the most widely consumed psychoactive substance, produces complex pharmacological actions; it has been shown to be chemopreventive in non-melamona skin cancer in mice through increasing apoptosis. Here we have investigated the molecular and cellular mechanisms in the pro-apoptotic effect of caffeine on UVB-irradiated human HaCaT keratinocytes. Pretreatment with caffeine increased UVB-induced apoptosis in HaCaT cells. Caffeine blocked UVB-induced Chk1 phosphorylation. In addition, similar to the effect of the PI3K inhibitor LY294002, caffeine also inhibited phosphorylation of AKT and up-regulation of COX-2, two critical oncogenic pathways in skin tumorigenesis. However, phosphorylation of EGFR or ERK was unaffected. Inhibiting ATR pathways by siRNA targeting ATR had little effect on UVB-induced apoptosis or AKT activation, indicating that the inhibitory effect of caffeine on apoptosis and the AKT pathway does not require the ATR pathway. Inhibiting AKT by caffeine blocked UVB-induced COX-2 up-regulation. Expression of constitutively active AKT that was not inhibited by caffeine was found to protect cells from caffeine-promoted apoptosis post-UVB irradiation, indicating that AKT is an essential inhibitory target for caffeine to promote apoptosis. Caffeine specifically sensitized cells with unrepaired DNA damage to UVB-induced apoptosis. These findings indicate that in HaCaT keratinocytes, inhibiting the AKT/COX-2 pathways through an ATR-independent pathway is a critical molecular mechanism by which caffeine promotes UVB-induced apoptosis of unrepaired keratinocytes for elimination.

Inhibitory effect of caffeine on pacemaker activity in the oviduct is mediated by cAMP‐regulated conductances

Spontaneous electrical activity, termed slow waves, drives rhythmic, propulsive contractions in the smooth muscle of the oviduct (myosalpinx). Myosalpinx contractions cause egg transport through the oviduct. Agents that disrupt slow wave pacemaker activity will therefore disrupt myosalpinx contractions and egg transport. Caffeine is commonly used as a ryanodine receptor agonist and has been previously associated with delayed conception. Here we assessed the effects of caffeine on pacemaker activity in the murine myosalpinx. The effects of caffeine on electrical pacemaker activity were studied using intracellular microelectrode and isometric force measurements on intact oviduct muscle preparations. Responses to caffeine were compared with responses caused by 3-isobutyl-1-methylxanthine (IBMX) and forskolin. Caffeine caused hyperpolarization of membrane potential and inhibited slow wave generation and myosalpinx contractions. The effects of caffeine could be mimicked by the K(ATP) channel agonist pinacidil and antagonized by the K(ATP) channel antagonist glibenclamide. Caffeine is known to inhibit cyclic nucleotide phosphodiesterases (PDEs), leading to an increase in cytosolic cAMP and stimulation of downstream cAMP-dependent mechanisms. The effects of caffeine were mimicked by the PDE inhibitor, IBMX, and the adenylyl cyclase activator forskolin. These effects were also reversed by glibenclamide. These results suggest that caffeine activates K(ATP) channels in oviduct myosalpinx. Since caffeine abolishes slow waves and associated contractions of the myosalpinx, it would have a negative effect on egg transport through the oviduct and may contribute to the documented delayed conception in women consuming caffeinated beverages.

Long‐term high calcium decreases AMP‐activated protein kinase (AMPK) activity via activating protein phosphatase 2A

AMP‐activated protein kinase (AMPK) is activated by upstream kinases and negatively regulated by protein phosphatase 2A (PP2A). Calcium has recently been found to activate PP2A, which contains calcium binding sites. Our previous work has shown that chronic calcium exposure decreases AMPK activity. To define the specific molecular mechanism, activity of AMPK and PP2A was analyzed in C2C12 and skeletal muscles from mutant pigs possessing AMPKδ3 R225Q, ryanodine receptor mutation, or both. C2C12 myotubes treated with caffeine (calcium releasing agent) for 10 h decreased AICAR‐induced AMPK activity to control level and this negative effect has diminished by dantrolene (ryanodine receptor stabilizer). Interestingly, muscles from pigs with ryanodine receptor mutation and C2C12 cells administered with 10 h caffeine showed higher PP2A activity compared to control (P &lt; 0.05). More importantly, the inhibitory effect of caffeine on AMPK activity was attenuated by PP2A inhibitor, okadaic acid or PP2A RNA interfering. These data show the inhibitory effect of chronic calcium on AMPK activity is exerted through the activation of PP2A.

Inhibitory effects of caffeine and its metabolites on intracellular lipid accumulation in murine 3T3-L1 adipocytes.

To understand the mechanisms of the anti-obesity effects of dietary caffeine, the effects of caffeine and its metabolites on adipocyte differentiation and insulin-stimulated glucose uptake in murine 3T3-L1 adipocytes were investigated. Caffeine did not inhibit the differentiation of 3T3-L1 pre-adipocytes to mature adipocytes, but it did suppress the intracellular lipid accumulation after complete differentiation in a dose-dependent manner (0.125-1.0 mM). This effect was also observed in 1,3,7-trimethyluric acid- 3,7-dimethyluric acid- and 5-acetylamino-6-formylamino-3-methyluracil-treated cells. Caffeine also inhibited insulin-stimulated glucose uptake in differentiated 3T3-L1 adipocytes in a dose-dependent manner. Treatment with theophylline, paraxanthine, 1-methylxanthine (MX), 3-MX, or 7-MX also inhibited glucose uptake in differentiated adipocytes. These results suggest that the anti-obesity activity of dietary caffeine is due to the additive and/or synergistic inhibitory effects of caffeine and its metabolites on intracellular lipid accumulation and that caffeine does not affect adipocyte differentiation.

Inhibitory Effect of Caffeine on Growth of Various Bacterial Strains

Inhibitory Effect of Caffeine on Growth of Various Bacterial Strains

The adenosine A 1 receptor contributes to the stimulatory, but not the inhibitory effect of caffeine on locomotion: a study in mice lacking adenosine A 1 and/or A 2A receptors

Caffeine has biphasic effects on locomotion, and blockade of the adenosine A 2A receptor (A2AR) is necessary for the stimulatory effect of low doses of caffeine, but not for the locomotor depressant effect observed at high doses. We wanted to elucidate the role of the adenosine A 1 receptor (A1R) in mediating the locomotor effects of increasing doses of caffeine using wild-type mice (A1R WT), mice heterozygous for (A1R HET), and mice lacking the adenosine A 1 receptor (A1R KO). Caffeine had the typical biphasic dose–effect relationship in all three genotypes, but the stimulatory action of caffeine was facilitated in the A1R KO mice. In order to investigate the interaction between blockade of A1Rs and A2ARs, mice lacking both receptors (A1R KO/A2AR KO) were tested. Regardless of A1R genotype, animals lacking A2AR were not stimulated by caffeine, whereas animals heterozygous for A2AR were. As expected, the A1R is not crucial for the stimulatory effect of caffeine, but seems to modulate the effect of caffeine exerted via A2AR blockade. Furthermore, these results suggest that the inhibitory effect of high doses of caffeine is due neither to blockade of the A1R, nor of the A2AR, and an effect independent of these adenosine receptors is likely.