(Circulation. 2000;102:285.)
© 2000 American Heart Association, Inc.
Clinical Investigation and Reports |
Dose and Time Effects of Caffeine Intake on Human Platelet Adenosine A2A Receptors
Functional and Biochemical Aspects
From the Department of Clinical and Experimental Medicine, University of Ferrara (K.V., F.P., S.G., S.M., P.A.B.), and Schering-Plough Research Institute, Milan (E.O.), Italy; and Department of Medicine, Division of Cardiology, University of Florida, Gainesville (L.B.).
Correspondence to Prof Pier Andrea Borea, Department of Clinical and Experimental Medicine, University of Ferrara, Via Fossato di Mortara 17-19, 44100 Ferrara, Italy. E-mail bpa{at}dns.unife.it
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Abstract |
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Background—We determined whether repeated caffeine administration at different dosages and for different periods of time (400 or 600 mg/d for 1 week or 400 mg/d for 2 weeks) upregulates human platelet adenosine A2A receptors and is accompanied by increases in cAMP accumulation and decreases in aggregation and calcium levels after stimulation of A2A receptors by the selective agonist 2-hexynyl-5'-N-ethylcarboxamidoadenosine (HE-NECA).
Methods and Results—Platelets were obtained from peripheral venous blood of 45 healthy human volunteers at the end of 2 weeks of caffeine abstinence and at 12, 60, and 108 hours after the last dose of caffeine. The lowest dose of caffeine, when given for only 7 days, had no effect. Increasing the total dose, either by giving 400 mg/d for 14 days or giving 600 mg/d, resulted in binding assays performed with the adenosine A2A receptor radioligand [3H]SCH 58261 [5-amino-7(phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine], in the upregulation of A2A receptors. Moreover, the potency of HE-NECA to produce antiaggregatory effects, a rise in cAMP accumulation, and a decrease in calcium levels was significantly increased.
Conclusions—Chronic caffeine intake can lead to upregulation of adenosine A2A receptors, which is accompanied by sensitization, in a time- and dose-dependent manner, to the actions of the agonist HE-NECA.
Key Words: adenosine • receptors • caffeine • platelets
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Introduction |
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Caffeine, found in different sources such as coffee, tea, chocolate, and cola drinks, is the most widely active substance in the world. Average caffeine consumption by adult humans varies among different cultures and nations from 80 to 400 mg per person per day.1 Caffeine elicits a diverse number of pharmacological responses, including increased vigilance, decreased psychomotor reaction time, and increased sleep latency and waking time and may also influence intellectual performance.2 Moreover, caffeine causes relaxation of smooth muscles, enhances the secretion of gastric acid and the release of catecholamines, and increases metabolic activity.3
The precise mechanism(s) underlying the actions of caffeine remain poorly defined. Although the inhibition of phosphodiesterases may contribute to the actions of caffeine,4 there is growing evidence that most pharmacological effects of this xanthine result from antagonism of adenosine receptors designated as A1, A2A, A2B, and A3 subtypes.5 Caffeine acts most potently at A2A receptors, followed closely by A1 receptors, then A2B receptors,6 and as a weak antagonist at human A3 receptors. Blockade by caffeine of adenosine receptors, namely the A1 and the A2A receptor types, inhibits the action of endogenous adenosine on a variety of physiological processes.7 Under normal conditions, blood levels of adenosine appear to be sufficient to tonically activate A2A receptors in platelets. Recently, in A2A receptor–knockout mice, it was reported that platelet aggregation was increased, indicating the importance of this receptor subtype in platelet function.8 It is therefore conceivable that caffeine could block these tonically activated A2A receptors in platelets and alter their functions modulated by adenosine.
For many years, an association has been suspected between coffee drinking and cardiovascular diseases, in particular coronary heart disease,9 but recently it has been demonstrated that coffee or caffeine consumption does not increase the risk of coronary heart diseases or stroke.10 11 Numerous epidemiological studies considering myocardial infarction have found no deleterious effect of <5 cups of coffee per day, whereas results are controversial at higher intake levels.12 In patients with hypertension, no adverse outcome risk was observed at any level of caffeine intake.13
A study by Biaggioni et al14 found that a repeated dosing regimen of caffeine leads to significant changes in human platelets in the functional responses to the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA). Caffeine withdrawal produced a significant leftward shift of the NECA-induced inhibition of aggregation.14 Consistent with this, we recently demonstrated,15 in subjects treated with 750 mg/d for 1 week, that chronic intake of caffeine alters the platelet aggregability as a result of upregulation of the A2A receptors located on the platelet surface.
In the present article, we provide further evidence that a similar response is found in subjects treated with caffeine at different doses, such as 600 mg/d for 1 week or 400 mg/d, administered for a longer period of time, such as 2 weeks. In the present study, this was done by directly measuring adenosine A2A receptor changes (density and affinity) and their function by determining the effect of the A2A selective agonist 2-hexynyl-NECA (HE-NECA) to (1) increase cAMP accumulation, (2) inhibit platelet aggregation, and (3) decrease intracellular calcium levels. After chronic consumption (600 mg/d for 1 week or 400 mg/d for 2 weeks), upregulation of platelet adenosine A2A receptors was found, which was highly correlated with antiaggregatory effects, a rise in cAMP accumulation, and a decrease in intracellular calcium levels. No differences were revealed on binding and functional parameters from subjects treated with 400 mg/d for 1 week.
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Methods |
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Forty-five healthy, nonsmoking subjects, 25 to 45 years of age, of both sexes, were studied. After having given written informed consent, subjects were asked to abstain from dietary methylxanthines for
2 weeks. They were divided into 3 groups (15 subjects each)
according to the caffeine administration regimen (ie, dose and duration
of administration): 200 mg orally 2 times a day for a period of 7 days
(group 1); 200 mg orally 2 times a day for a period of 14 days (group
2); and 200 mg orally 3 times a day for a period of 7 days (group
3). Platelets from these subjects were studied before they
started caffeine (day 0) and at 1, 12, 60, and 108 hours after the last
dose of caffeine. In particular, the 1-hour time point was studied only
in the group receiving the maximum dosage of caffeine (600 mg/d).
EC50 and IC50 in functional
assays could not be obtained at 1 hour because of the practical
problems linked to excessive drawing of blood in a narrow period of
time (1 to 12 hours).
[3H]SCH 58261 Binding Assay in Human Platelet
Membranes
Membranes from human platelets were prepared as previously
described16 and used for radioligand binding
assays with [3H]SCH 58261 according to Varani
et al.17 In saturation studies, human platelet
membranes were incubated with 8 to 10 different concentrations of
[3H]SCH 58261 ranging from 0.01 to 10 nmol/L.
Nonspecific binding was determined in the presence of NECA 10
µmol/L. After 60 minutes of incubation at 4°C, samples were
filtered through Whatman GF/B filters with a Micro-Mate 196 Cell
Harvester (Packard Instrument Co). A weighted nonlinear least-squares
curve-fitting program, LIGAND,18 was used for computer
analysis of the data from the saturation experiments.
Measurement of cAMP Levels in Human Platelets
Washed human platelets obtained from the
peripheral blood of healthy volunteers were prepared as
described previously.16 Platelets
(6x104 to 8x104 cells)
were incubated with 1.0 U of adenosine deaminase/mL, 0.5
mmol/L 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724) as
phosphodiesterase inhibitor, and 6 to 8 different
concentrations of HE-NECA. EC50 values were
obtained from concentration-response curves after log-logit
transformation of dependent variables by a weighted least-squares
method.19 The final aqueous solution was tested for cAMP
levels by a competition protein binding assay.15
Platelet Aggregation Assay
Citrated human blood was centrifuged at 200g
for 10 minutes to obtain platelet-rich plasma and at
2500g for 20 minutes to obtain platelet-poor plasma.
Human platelet counts were performed in a Coulter Counter model
S8/80 (Coulter Electronics Inc) and adjusted to
2.5x108/mL to 3.5x108/mL
with autologous platelet-poor plasma. Platelet aggregation was
performed according to the Born turbidimetric
technique20 with a DIC PA-3220 Aggrecorder (Kyoto
Daiichi Kagatu Co). After incubation for 3 minutes with 6 to 8
different concentrations of HE-NECA, platelet-rich plasma was
aggregated at 37°C under continuous stirring with ADP. Similar
experiments were performed with ADP at different concentrations (100
nmol/L to 100 µmol/L). Maximal aggregation, recorded 5
minutes after the addition of ADP, was used for the quantitative
analysis, and the percentage of inhibition was calculated in
relation to control values.21
Cytoplasmic Free Ca2+ Concentration
Measurements
Cytosolic free calcium concentration was measured by use of the
fura 2 technique according to Paul et al.22
Briefly, platelets were incubated in total darkness for 30 minutes
at 37°C with 1 µmol/L fura 2-AM and magnetically stirred in
fluorimeter cuvettes (LS50, Perkin Elmer, Ltd) at a concentration of
108/mL in the presence of 250 µmol/L
sulfinpyrazone. Intracellular Ca2+ concentration
([Ca2+]i) was determined
at an excitation ratio of 340/380 and an emission wavelength of 505
nm.
Statistical Analysis
Analysis of data was carried out by 1-way ANOVA.
Analysis of difference between caffeine-treated groups (12, 60,
and 108 hours) and control subjects was done with Student’s
t test (unpaired analysis). Differences were
considered significant at a value of P<0.01. All data are
reported as mean±SEM.
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Results |
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Platelets from the 3 groups of subjects were harvested before the administration of caffeine was begun (day 0, control) and at 1, 12, 60, and 108 hours after the last dose (caffeine withdrawal). The Table
summarizes the results of
binding and functional experiments from the 3 groups of subjects.
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Group 1 (400 mg/d for 1 Week)
Binding parameters revealed a control
Bmax value of 105±6 fmol/mg protein and a
KD value of 1.28±0.08 nmol/L. In control
platelets, HE-NECA increased cAMP levels with an
EC50 of 60±5 nmol/L and inhibited (1)
aggregation with an IC50 of 86±10 nmol/L and (2)
calcium levels with an IC50 of 104±8 nmol/L. No
statistically significant differences were found in binding and
functional parameters at 12, 60, and 108 hours after
caffeine withdrawal (Table
).
Group 2 (400 mg/d for 2 Weeks)
Binding parameters revealed a control
Bmax value of 110±3 fmol/mg protein and a
KD value of 1.21±0.09 nmol/L. At 12 and 60
hours after caffeine withdrawal, the receptor densities,
Bmax, increased at both time points by some 20%,
but the KD values were unchanged.
Functional experiments revealed that the adenosine
A2A receptor agonist HE-NECA was significantly
more potent in increasing platelet cAMP (ie, the
EC50 values were 45% and 65% smaller at 12 and
60 hours after caffeine withdrawal than control values, respectively).
A similar trend was observed in the IC50 values
obtained in aggregation experiments and in cytoplasmic free calcium
concentration measurements (Table).
Group 3 (600 mg/d for 1 Week)
Overall, we obtained data similar to those of group 2.
[3H]SCH 58261 bound to a single affinity class
of sites in platelet membranes from controls with a
Bmax of 100±4 fmol/mg protein and a
KD of 1.27±0.09 nmol/L. As shown in Figure 1A, in membranes from platelets
harvested at 1, 12, 60, and 108 hours after caffeine withdrawal, the
radioligand bound with the same affinity, but the number of
binding sites (Bmax) was significantly
(P<0.01) increased. In parallel studies, the functional
responses of platelets to the A2A receptor
agonist HE-NECA were determined. As summarized in the Table, the
potency of HE-NECA to (1) increase cAMP formation, (2) inhibit
ADP-induced platelet aggregation, and (3) decrease calcium levels
was significantly increased in platelets obtained at 12, 60, and
108 hours after caffeine withdrawal (Figure 1B, 1C, and 1D).
Experiments were also carried out to determine whether the increase in
the density of A2A receptors would be accompanied
by a decrease in potency and/or efficiency of ADP to induce
aggregation. The EC50 values of ADP to stimulate
platelet aggregation at 12, 60, and 108 hours after caffeine
withdrawal were 0.7±0.2, 0.9±0.1, and 0.8±0.1 µmol/L,
respectively, values not significantly different from the 0.9±0.2
µmol/L obtained in platelets from controls (Figure 2).
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) and 12
(•), 60 (
), and 108 (
) hours after caffeine withdrawal. Inset,
Scatchard plot of specific binding. B and F denote bound and free
ligand, respectively. B, C, and D, HE-NECA concentration-effect curves
to stimulate accumulation of platelet cAMP (B), to inhibit
platelet aggregation (C), and to inhibit calcium levels (D).
Platelets were obtained from human subjects before caffeine
administration (control
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Discussion |
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The effects of long-term administration of caffeine in humans and animals and its role in their tolerance to the actions of caffeine are controversial. Some studies showing an increase of A1 receptors in the mouse brain found evidence for a dose-dependent upregulation of A1 receptors by caffeine.23 Other studies revealed an upregulation of A2A receptors, suggesting an adaptive effect of caffeine intake.24 Moreover, chronic caffeine consumption may lead to a reduction in platelet aggregability as a result of upregulation of the A2A receptors located on the platelet surface14 playing a potential role in pathophysiological processes, such as aggregation and thrombogenesis. Although such changes may contribute to alterations in platelet function, tolerance to caffeine does not modify the potency of this xanthine as a competitive antagonist of the effects of adenosine.25 Other changes, such as shifting of receptor affinity to a high-affinity state, alterations in G protein levels or the coupling of these proteins to adenosine receptors, or long-term receptor occupancy may be involved in the phenomenon of tolerance.26 Withdrawal symptoms to caffeine occur in humans; typically, they are headache, fatigue, apathy, and drowsiness.9 In particular, caffeine increases plasma adenosine concentration, and its reduction after antagonist withdrawal suggests receptor-mediated regulation of the plasma adenosine concentration.27 During ischemia and/or hypoxia, adenosine also has neuroprotective actions. In the adult brain, chronic caffeine treatment, which leads to upregulation of adenosine receptors, reduces ischemic damage, whereas acute exposure (receptor antagonistic effect) increases ischemic damage.28
It has been demonstrated that chronic intake of caffeine alters the response of platelets to the actions of adenosine.14 Repeated administration of caffeine (750 mg/d for 1 week) revealed an increase in A2A receptor density, accompanied by sensitization of platelet responses, such as an increase in cAMP accumulation and decrease in platelet aggregation.15 The aim of the present study was to determine the effect of caffeine dosage and of the duration of administration on binding and functional parameters. Hence, we studied the changes in the density and affinity of adenosine A2A receptors in human platelet membranes of subjects treated with different doses (400 or 600 mg/d) for different periods of caffeine intake (1 or 2 weeks). Specifically, we studied control (before caffeine administration) and caffeine-treated (1, 12, 60, and 108 hours after the last dose of caffeine) subjects.
The treatment with 400 mg/d caffeine for 1 week did not modify A2A receptor binding and functional parameters. However, treatment with 400 mg/d for 2 weeks or 600 mg/d for 1 week resulted in (1) a significant increase (upregulation) of adenosine A2A binding sites, (2) a rise in cAMP accumulation, (3) an increase of antiaggregatory effects, and (4) a decrease in calcium levels elicited by the A2A receptor agonist HE-NECA.
The upregulation of A2A receptors can probably be ascribed to the synthesis of new receptors during differentiation of precursor cells. This interpretation is based on the results of in vitro experiments showing that the incubation of platelet-rich plasma from control subjects for a period of 6 or 12 hours with caffeine or SCH 58261 did not affect the binding parameters.15 The upregulation of adenosine A2A receptors caused by chronic intake of caffeine could be interpreted to indicate that endogenous adenosine has a tonic influence on human platelets, and the presence of the antagonist is counterbalanced by the upregulation of A2A receptors. In adults, caffeine is adsorbed efficiently from the gastrointestinal tract; peak plasma concentrations occur 15 to 120 minutes after ingestion, and the half-life of caffeine is 2.5 to 4.5 hours.7 The increase of adenosine A2A receptors found at 1 hour after the last dose of caffeine treatment was similar to that obtained at 12 or 60 hours after caffeine withdrawal, showing that the withdrawal was not necessary for the upregulation of A2A receptors.
Another aim of the present study was to determine whether the changes in binding parameters correlated with changes in functional response(s). It was found that platelet aggregation was associated with activation of adenylate cyclase and with a rise in intracellular calcium concentrations. The potency of HE-NECA at 12, 60, and 108 hours after caffeine withdrawal was significantly increased compared with the control group. This finding indicates that repeated administration of different doses of caffeine leads to significant changes in the number of A2A receptors on the platelet surface, accompanied by an enhanced responsiveness to receptor stimulation. The classic adenosine A2A receptor is responsible for the antiaggregatory properties of adenosine and its analogues, which is consistent with the observation that aggregation is more efficient in mice lacking the A2A receptors.8 However, platelet aggregation induced by increasing concentrations of ADP was not significantly different between control and caffeine-treated subjects. One possible explanation for this latter observation is that there is not enough adenosine in the assay medium to produce a response. Alternatively, in the caffeine-treated subjects, the magnitude of receptor upregulation (ie, number of receptors) was not sufficient to produce a shift of the ADP-induced aggregation concentration-response curve. However, when the levels of endogenous adenosine increase, such as during myocardial ischemia, the extracellular concentration of adenosine rises rapidly to a level sufficient to act on upregulated receptors and may have greater platelet-inhibitory effects than control. Thus, it is possible that chronic caffeine consumption in doses not far from the average dietary intake may lead to a paradoxical reduction in platelet aggregability during ischemia.
In conclusion, all the data together provide further evidence that chronic intake of caffeine alters the response of platelets to the actions of adenosine. The major finding of the present study is that the effects of chronic caffeine consumption on platelet functions are dependent on both the dose and the duration of the treatment and underlie a reduction in platelet aggregability due to upregulation of the adenosine A2A receptors.
Received November 1, 1999; revision received January 21, 2000; accepted February 15, 2000.
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