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. Author manuscript; available in PMC: 2011 Jun 1.
Published in final edited form as: Acta Physiol (Oxf). 2010 Feb 1;199(2):221–230. doi: 10.1111/j.1748-1716.2010.02091.x

Adenosine A3 receptors regulate heart rate, motor activity and body temperature

Jiangning Yang 1,*, Yingqing Wang 1,2,*, Pablo Garcia-Roves 1, Marie Björnholm 1,3, Bertil B Fredholm 1,†
PMCID: PMC2883671  NIHMSID: NIHMS182862  PMID: 20121716

Abstract

Aim

We wanted to examine the phenotype of mice that lack the adenosine A3 receptor (A3R).

Methods

We examined the heart rate, body temperature and locomotion continuously by telemetry over several days. In addition the effect of the adenosine analogue R - N6- phenylisopropyl-adenosine (R-PIA) was examined. In addition, we examined heat production and food intake.

Results

We found that the marked diurnal variation in activity, heart rate and body temperature, with markedly higher values at night than during day time, was reduced in the A3R knockout mice. Surprisingly, the reduction in heart rate, activity and body temperature seen after injection of R-PIA in wild type mice was virtually eliminated in the A3R knock-out mice. The marked reduction in activity was associated with a decreased heat production, as expected. However, the A3R knock-out mice, surprisingly, had a higher food intake but no difference in body weight compared to wild type mice.

Conclusions

The mice lacking adenosine A3 receptors exhibit a surprisingly clear phenotype with changes in e.g. diurnal rhythm and temperature regulation. Whether these effects are due to a physiological role of A3 receptors in these processes or if they represent a role in development remains to be elucidated.

Keywords: Sex differences, heat production, body weight, diurnal, knock-out

INTRODUCTION

Of the four adenosine receptors, the A3 receptor was the one most recently identified by cloning (Zhou et al. 1992). This receptor is often described as being “low affinity” (e.g. Bozarov et al. 2009) but there is evidence that at comparable receptor densities adenosine is as good an agonist at the A3 receptor as it is on A1 and A2A receptors (Fredholm et al. 2001). Interestingly, inosine is a partial agonist at the A3 receptor and this adenosine metabolite appears to mediate some responses via A3 receptors (Jin et al. 1997; Gomez & Sitkovsky 2003). The late discovery and the fact that there are major species differences in structure and pharmacology hampered the elucidation of its functional role(s) by pharmacological methods. The development of a knock-out mouse (Salvatore et al. 2000) has therefore been very useful to delineate physiological and pathophysiological situations where A3 receptors are important (Yaar et al. 2005). In many ways the study of genetically modified mice has stimulated the interest in adenosine A3 receptor pharmacology (Borea et al. 2009).

Already the first study on the knock-out mice emphasized that A3 receptors are important in modulating responses of inflammatory cells (Salvatore et al. 2000). Activation of A3 receptors enhances responses of mast cells (Jin et al. 1997; Salvatore et al. 2000; Zhong et al. 2003), which can further lead to increased vascular permeability (Tilley et al. 2000), inflammatory pain (Wu et al. 2002) as well as increased bronchoconstriction (Tilley et al. 2003; Hua et al. 2008). On the other hand, there can be an A3 receptor-mediated reduction of the release of TNFα (Salvatore et al. 2000), and methotrexate, by increasing adenosine levels, appears to utilize this mechanism to reduce TNFα responses during inflammation (Montesinos et al. 2003). However, the role of A3 receptors is less important than A2A receptors in several circumstances (Kreckler et al. 2006; Montesinos et al. 2006). Adenosine A3 receptors stimulate the migration of eosinophils (Young et al. 2004) and neutrophils (Chen et al. 2006b; Inoue et al. 2008; van der Hoeven et al. 2008).

There are also vascular effects. Although basal blood pressure is not altered in A3R (−/−) mice the blood pressure fall after adenosine was enhanced (Zhao et al. 2000), and the coronary flow after adenosine A2A receptor stimulation is higher in the A3R knock-out mouse (Talukder et al. 2002). There are also A3 receptors that mediate the ability of adenosine to, via changes to the glycocalyx, increase the vascular permeability (Platts & Duling 2004).

Elimination of A3 receptors appears to increase resistance to ischemic damage in the heart (Cerniway et al. 2001; Guo et al. 2001), and the negative effect of pressure overloading (Lu et al. 2008) but does not alter early preconditioning (Guo et al. 2001; Eckle et al. 2007). Paradoxically activation of A3 receptors also has been reported to confer resistance to myocardial ischemic damage (Harrison et al. 2002; Ge et al. 2006), and this occurs via activation of sarcolemmal kATP channels (Wan et al. 2008). Ischemic kidney damage is similarly reduced in mice lacking A3 receptors (Lee et al. 2003) whereas ischemic brain damage is increased (Chen et al. 2006a), as is skeletal muscle ischemia (Zheng et al. 2007). Tissue damage following sepsis is reduced by endogenous A3 receptor activation (Lee et al. 2006). Although the expression of A3 receptor is restricted and occurs late during development the over expression leads to embryonic lethality (Zhao et al. 2002). It was recently reported that the hepatic glucose mobilization after ischemia/reperfusion was virtually completely eliminated by an antagonist of adenosine A3 receptors (Cortés et al. 2009).

The behavioural consequences of an A3 receptor deletion are complex: Basal locomotor activity is increased (Fedorova et al. 2003; Björklund et al. 2008a), the activity is reduced in tests assumed to predicted changes in mood (Fedorova et al. 2003), and the behavioural activation by caffeine and amphetamine was reduced as was the natural increase in activity when the animals enter the dark night time (Björklund et al. 2008a). The expression of A3 receptors in brain is so limited that a role has been questioned (Rivkees et al. 2000), but it is clear that microglial cells and astrocytes possess functional A3 receptors (Hammarberg et al. 2003; Björklund et al. 2008c). The presence in nerve cells is supported by the finding that apparently selective A3 receptor activation increases serotonin transport in synaptosomes (Zhu et al. 2007) and by effects on long term potentiation (LTP, Costenla et al. 2001), which are eliminated in A3R knock-out mice (Maggi et al. 2009).

Thus, there are several indications that A3 receptors are involved in several physiological and pathophysiological reactions. However, there is little data where unrestrained mice have been followed for longer periods of time with respect to basic physiological parameters. Here we report initial findings using such methodology following up on earlier studies where mice lacking A1 and/or A2A receptors have been examined (Yang et al. 2007, 2009a-b). We confirm that mice lacking A3 receptors show clear alterations in activity (Björklund et al. 2008a), but we also demonstrate an unexpected role in temperature regulation and metabolism.

MATERIALS AND METHODS

Animals

The study was performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US NIH, and was approved by the Animal Ethics Committee of Northern Stockholm. We used adenosine A3 receptor deficient (A3R −/−) mice generated by Merck Research Laboratories (with permission from Drs Marlene Jacobson and Stephen Tilley) and wild type control mice (C57Bl/6). To minimize genetic drift there was frequent interbreeding to heterozygotes. The genotyping of A3R−/− mice was performed via using PCR. The mice were backcrossed repeatedly against C57Bl6 (more than 10 generations) to ensure that they were practically congenic.

The telemetry system

As previously described (Yang 2009), we used a telemetry system (Data Sciences, St. Paul, MN) consistingof implantable transmitters (TA10ETA-F20), telemetry receivers (DSI PhysioTel Receivers-RPC-1 Model), and eight universal adapters (UA 10 PC). Anaesthesia was induced and maintained by isoflurane. The transmitter was implanted into the peritoneal cavity and the leads were sutured in a lead II position. Mice were allowed to recover at least 7 days before the recordings of each individual mouse, kept in its own cage, were started. A computer program(Data quest A.R.T gold acquisition) sampled calibrated values of heart rate and body temperature as well as uncalibrated activity counts. After 5 days of baseline registration some of the mice received the adenosine analogue R(−)N6-(2-phenylisopropyl) adenosine (R-PIA, 50 μg/kg, Sigma, i.p.).

Whole Body Energy Homeostasis

Food intake, oxygen consumption, and locomotor activity were measured using a Comprehensive Lab Animal Monitoring System (Columbus Instruments, Columbus, OH). Mice were housed individually with ad libitum access to standard chow and water. Mice were acclimatized to the metabolic cages for 48 h prior to a 24-h period of automated recordings every 25 min. Oxygen consumption (VO2) was determined by an open-circuit Oxymax. Oxygen consumption was calculated as the difference between the input oxygen flow and the output oxygen flow. The system will also calculate carbon dioxide production (VCO2) and the respiratory exchange ratio (RER) as the ratio between VCO2 and VO2. The rate of heat production is calculated by first assessing the calorific value (CV) of the food being metabolized. The CV is based on the observed RER in a manner tabulated by Lusk (1928). The equations are: Heat = CV× VO2 − CV = 3.815 +1.232×RER and from this heat production was calculated. Ambulatory locomotor activity was measured by consecutive beam breaks in adjacent beams under the 24 h period.

Statistical analysis

Student’s t-test or ANOVA were employed for evaluation of differences between groups, using the procedures in GraphPad Prism.

RESULTS

Heart rate

Basal heart rate differed between male and female mice, as reported earlier (Yang et al. 2007). In male mice there was little difference between A3R +/+ and A3R −/− mice (Fig 1). By contrast, there was a major difference between genotypes in the females. Whereas female A3R +/+ mice showed the expected large diurnal variation, with higher rates at night, especially in the beginning, the heart rate curve was essentially flat in the A3R −/− mice (Fig 1). Another major surprise was that the well known cardio depressant effect of the adenosine analogue R-PIA, which is commonly ascribed to an action on cardiac A1 receptors (Johansson et al. 2001; Yang et al. 2007), was virtually eliminated in the A3R −/− mice (Fig 2). This was true for male as well as female mice.

Figure 1.

Figure 1

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Registrations of heart rate (HR) in male [wild type (WT), n=9; A3R knockout (A3R−/−), n=13; shown in panel A and female (WT, n=13; A3R−/−, n= 13; shown in panel B) mice. The dark period (shadowed) is from 7 PM to 7 AM. Data are shown as a 5-day average and at 10 min intervals; data are presented as mean values ± SE. The indicated p- value refers to the result of a 2-way ANOVA.

Figure 2.

Figure 2

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Effects of R(−)N6-(2-phenylisopropyl) adenosine (R-PIA, 50 μg/kg) on heart rate in A3R−/− mice. R-PIA was administrated into male [wild type (WT), n=5; A3 adenosine receptor knockout (A3R−/−), n=9] and female (WT, n=10; A3R−/−, n=5) via intra peritoneum. The heart rate was measured at 10-min intervals until 240 min after injection. Data are presented as means ± SE (*compared with WT, P < 0.05). Abbreviations: bpm, Beats/min.

Body temperature

The registration of body temperature generated results that were very similar to those for heart rate, as reported earlier in other genotypes (Yang et al. 2007). Temperature was somewhat higher in females, than in males, and the diurnal variation normally seen in female mice was absent in the mice lacking A3R receptors (Fig 3). Furthermore, the substansial decrease in body temperature induced by R-PIA was essentially eliminated in the A3R −/− mice of both sexes (Fig 4).

Figure 3.

Figure 3

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Registrations of body temperature for consecutive 10-min periods in male [wild type (WT), n=9; A3R knockout (A3−/−), n=13]; shown in panel A and female (WT, n=13; A3−/−, n= 13) mice; shown in panel B. The dark period (shadowed) is from 7 PM to 7 AM. Data are shown as a 5-day average; data are presented as mean values ± SE (*p < 0.05 compared with WT mice). The significance value refers to results of a 2-way ANOVA.

Figure 4.

Figure 4

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Effects of R(−)N6-(2-phenylisopropyl) adenosine (R-PIA, 50 μg/kg i.p.) on body temperature in A3−/− mice. R-PIA was administrated into male [wild type (WT), n=5; A3 adenosine receptor knockout (A3−/−), n=9] and female (WT, n=10; A3−/−, n=5). The body temperature was measured at 10-min intervals until 240 min after injection. Data are presented as means ± SE (*compared with WT, P < 0.05).

Locomotor activity

As expected the results from measurements of locomotion gave similar results. As reported earlier the increased over all activity seen in female mice when the light is about to turn off was essentially eliminated in the A3R −/− mice (Fig 5) (Björklund et al. 2008b). This was not seen to the same extent in the male mice studied with this technique (but see below). When animals were given an injection of R-PIA they initially were stimulated, but in the wild type animals this was followed by a long fall in activity both in males and females (Fig 6). In the A3R −/− mice, by contrast, there was no apparent depression of activity.

Figure 5.

Figure 5

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Baseline registrations of activity are recorded for consecutive 30-min periods in male [wild type (WT), n=9; A3R knockout (A3−/−), n=13; shown in panel A] and female (WT, n=13; A3−/−, n= 13; shown in panel B) mice. The dark period is from 7 PM to 7 AM. Data are shown as a 5-day average; data are presented as mean values ± SE (*p < 0.05 compared with WT mice).

Figure 6.

Figure 6

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Effects of R(−)N6-(2-phenylisopropyl) adenosine (R-PIA, 50 μg/kg) on activity in male (Panel A] and A3−/− mice (Panel B). R-PIA was administrated into male [wild type (WT), n=5; A3 adenosine receptor knockout (A3−/−), n=9) and female (WT, n=10; A3−/−, n=5) via intra peritoneum. The activity was measured at 30-min intervals until 240 min after injection. Data are presented as means ± SE (*compared with WT, P < 0.05).

Locomotor activity could also be determined in the clams system. As seen in Fig 7 when this way of recording locomotion was used there appeared to be a clear-cut reduction in night time activity not only in females, but also in males (Fig 7, A, B, E), however in males it was not statistically significant if calculated as groups means. By contrast there were no significant reduction in day time activity (Fig 7D).

Figure 7.

Figure 7

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Locomotor activity differences between WT mice and A3−/− mice.

Panel A and Panel B show consecutive locomotor activities in 24 hr. Each point represents number of ambulatory activities during 25min (mean ± SE). Both in male (WT, n=6; A3−/−, n=7) and female (WT, n=4; A3−/−, n=6), A3−/− mice moved much less than WT mice, especially during the dark time, p<0.0001 (2 way ANOVA). Panel C and D are the activities in light and dark time separately. * p < 0.05.

Heat production

From measurements of oxygen consumption heat production can be calculated. As expected, given the decreased physical activity, the A3R −/− mice exhibited a lower heat production, especially at night (Fig 8). During the light period, A3R−/− mice produce 0.363±0.021 kcal heat in one hour, which is significantly, p<0.001, less than the WT mice (0.422±0.024 kcal/hr); in the dark, both genotypes had a higher heat production, but A3R−/− mice (0.420±0.021 kcal/hr) still produced significantly, p<0.0001, less than WT mice (0.513±0.024 kcal/hr).

Figure 8.

Figure 8

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Heat Production in male WT mice and A3−/− mice (WT, n=6; A3−/−, n=7). The figure shows calculated heat production, at 25 min intervals, over the 24 hr period. Each point represents mean ± SEM. WT mice produced significantly more heat (p < 0.001) than A3−/− mice when calculated over the entire period.

Food intake and weight gain

Given the lower activity and the lower heat production we expected to see a lower food intake as well. However, to our surprise, A3R −/− mice actually consumed more food than the corresponding wild types (Figure 9). During light time, on average, each male WT mouse consumed 0.073±0.001 gram (g) in one hour, and the A3R−/− mouse consumed almost 30 % more (0.109±0.015 g/hr); during the dark time, the male WT mouse consumed about twice as much (0.171±0.020 g/hr), and the A3R−/− mouse still consumed more (0.203±0.021 g/hr). Essentially similar data were obtained in females. The increased food intake in the face of lower heat production suggests a major increase in body weight. However, this was not observed. Instead the weight gain with age in A3R +/+ and A3R −/− mice, could be virtually superimposed, but with different curves for males and females (Figure 10).

Figure 9.

Figure 9

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Food intake in male WT and A3−/− mice. Panel A shows accumulated food intake in 24 hr (from noon the first day to noon the second day) of male mice (WT, n=6; A3−/−, n=7). A3−/− mice consumed much more food during 24hr than WT mice, p<0.0001 (2 way ANOVA). Each point represents accumulated food intake of every 25mins (mean ± SE).

Figure 10.

Figure 10

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Body weight of male and female wild type (WT) and A3 −/− mice with increasing age. Data are mean and S.D. Wild types males were 35, 15 and 17 in the different age groups, wild type females were 40, 10 and 19; A3 −/− males were 20, 8 and 16, and A3 −/− females were 45, 7 and 26 in the age brackets 2-3 months, 3-5 months and 5-9 months.

DISCUSSION

The present study has revealed several surprisingly strong phenotypes for the A3R −/− mouse, and there are a number of novel observations. The first major finding was that much of the effect of the purportedly A1 selective agonist R-PIA on heart rate, body temperature and activity was lost in the A3R −/− mouse. R-PIA was one of the first adenosine analogues used in the characterizations of adenosine receptors and was used in the initial definition of A1 receptors (Vapaatalo et al. 1975; van Calker et al. 1979; Londos et al. 1980). When it was shown that much (if not all) the cardiodepression and temperature lowering effect of R-PIA was lost in A1R −/− mice (Johansson et al. 2001; Yang et al. 2007) the case appeared settled. However, it has been known that R-PIA is also quite potent as a ligand at A3 receptors (Klotz et al. 1998). Our finding indicates that both A1 and A3 receptors are important. They also suggest that some of the responses hitherto solely ascribed to A1 receptors may actually also have a component dependent on A3 receptors. It is important to note that there is no change in the expression of A1 receptors in brains of A3R knock-out mice (Björklund et al. 2008b).

The second major finding is the disruption of the normal diurnal rhythm seen in especially female mice lacking the A3 receptor. We have earlier noted that the arousal induced by pharmacological agents such as caffeine and amphetamine is also reduced in these mice (Björklund et al. 2008b). This suggests that the A3 receptor is not specifically involved in regulation of diurnal rhythm, but rather that they play some role in arousal, irrespective of how it is induced. In the experiments with telemetry there appeared to be a very clear sex difference, but this was less clear-cut in the experiments using the CLAMS system. We have no good explanation for this apparent sex difference, but we note that the reduction in the effect of stimulatory drugs is seen in both sexes (Björklund et al. 2008b).

The third major finding relates to the surprising fact that the lower heat production was accompanied by increased food consumption. The decreased heat production was expected from the lower activity of the animals and we have no good reason to doubt this measurement. The food consumption is directly assessed by the system and again we have no good reason to doubt that measurement. A high food intake in the presence of decreased energy consumption/activity is obviously a recipe for considerable weight gain. However, we found no evidence for this in the A3R knock-out mice. It could be that the CLAMS system we used provides a completely artificial situation and the activity and food intake etc in the “normal” situation of the mice is quite different. We have little reason to assume that this is the case as for example activity measurements with telemetry gave at least somewhat similar results. An alternative possibility is that the absorption of nutrients is compromised in A3R knock-out mice and future work will try to test this possibility. We will also test if the body composition is altered even if the body weight is not.

Given that the A3 receptor was unknown for so long and that adenosine physiology and pharmacology were studied so long (without finding evidence for A3 receptors) the long list of phenotypes of the mice with a targeted A3 receptor gene deletion is surprising. In particular, quite a lot of the data indicate an altered basal physiology. Physiological adenosine levels are low (Zetterström et al. 1982; Lönnroth et al. 1989; Ballarín et al. 1991), and such concentrations are expected only to activate adenosine receptors where they are quite abundant (Fredholm et al. 2005). A3 receptors are generally not very abundant, and we are therefore left with a conundrum. This is amplified by the fact that often when a physiologically important mechanism is ablated genetically there are compensations. We certainly do not know the solution to this, but believe that one should consider the possibility that A3 receptors play an important role in development and that some of the phenotypes we observe are, in fact, consequences of such early developmental effects. We think it is possibly relevant in this context that, as indicated in the Introduction, A3 receptors do play a role in the migration of eosinophils and neutrophils(Young et al. 2004; Chen et al. 2006b; Inoue et al. 2008; van der Hoeven et al. 2008) and possibly other cells, and in the regulation of the extracellular matrix (Platts & Duling 2004). Irrespective of whether A3 receptors regulate physiology or regulate the development of the physiological systems, it is clear that much work is needed to further elucidate their roles.

Acknowledgements

The A3R −/− mice used in this study were originally provided by Marlene Jacobson and Stephen Tilley. We thank Eva Lindgren and Karin Lindström-Törnqvist for genotyping. Many of the experiments were carried out in the core facility for genetic physiology. We thank the Swedish science research council (grant no 2553), the Heart and Lung fund, NIH (Ro1 NS048995), Novo Nordisk fund for Endocrinological research, Hjärnfonden and Knut and Alice Wallenberg Foundation (2005.0120) for generous support.

Footnotes

Conflict of Interest.

There is no conflict of interest.

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