Studies Of The Adenosine Receptor And Adenosine Receptor Antagonist Caffeine Using Receptor Knockout Mice As Tools

Adenosine and its receptors have been broadly studied. However, the researches of the basic physiological roles of adenosine and its receptors are limited, for the lower selectivity of agonists and antagonists of adenosine receptors and there were no receptor gene knockout mice in a long period. In this study, we gave some new interpretations of the physiological roles of adenosine receptors by using adenosine receptor knockout mice with combination of selective adenosine receptor agonists and antagonists. Further, caffeine which is believed as an adenosine receptor antagonist and commonly applied in day life and clinical research was also studied. We tested the mechanism of high dose (75mg/kg) caffeine intake based on the biphasic effects caffeine showed.First, the effect of A₁ receptor deletion on blood pressure in mice was recorded by Samba blood pressure measuring system, and then the mice were given the nonselective adenosine receptor agonist (NECA) to see the effect of NECA on blood pressure. The effect of adenosine receptors on vascular activity was also measured by Danish Myotechnology wire myograph 610M system. Results showed blood pressure was much higher in adenosine A₁ receptor knockout (A₁R-/-) mice under anesthesia, but variability induced by NECA was unaltered. Augmentation index (AIX) of blood pressure was not different between A₁R-/- and wildtype (WT) mice, but the NECA-induced fall was larger in A₁R-/- mice. In vascular activity experiment, we found the contraction of aortic rings to phenylephrine or relaxations to acetylcholine were similar between different genotype mice. NECA enhanced the contractile response, and this was eliminated in aortas from A₁R-/- mice, incremental relaxation was seen in A₁R-/- mice aortic artery instead. However, in mesenteric arteries no contractile response was seen and adenosine-mediated relaxation was identical between studied genotypes. A2B adenosine receptors, rather than A2A receptors, may be mainly responsible for the vasorelaxation induced by adenosine analogues in the examined mice vessels. So we conclude that lack of adenosine A₁R could cause higher blood pressure in mice, the role of adenosine A₁ receptors in regulating vessel tone differs between blood vessels. Furthermore, contractile effects of A₁ receptor on isolated vessels cannot explain the blood pressure change in A₁R-/- mice. The A₁ receptor modulation of blood pressure is therefore mainly related to extravascular factors.Heart rate, body temperature and locomotion activity were continuously recorded by telemetric system over several days in free moving A₃ receptor deletion mice. In addition, the effect of adenosine A₁ receptor selective agonist R-N6-phenylisopropyl-adenosine (R-PIA) on A₃ receptor knockout (A₃R -/-) mice was examined. Heat production and food intake of A₃R -/- mice were also tested by comprehensive lab animal monitoring system (CLAMS). From the experiments, we found that the marked diurnal variation in activity, heart rate and body temperature which were markedly higher values at night than during day time, was reduced in the female A₃R -/- mice, but no significant difference in male mice. The reduction in heart rate, activity and body temperature seen after injection of R-PIA in WT mice was virtually eliminated in the A₃R-/- mice. The CLAMS data showed the marked reduction in activity was associated with a decreased heat production of A₃R -/- mice, as expected. However, the A₃R -/- mice had a higher food intake but no difference in body weight compared to WT mice. The fat composition was lower than the WT mice, and glucose tolerance was significantly improved in A₃R-/- mice, especially obvious in male mice. The data indicated that the mice lacking adenosine A₃ receptors exhibited a surprisingly clear phenotype with changes in diurnal rhythm particularly evident in female mice, but there is no good explaination for the sex induced difference. The inhibition effect of R-PIA was lost in A₃R -/- mice indicated both A₁ and A₃ receptors are important for R-PIA. Antagonizing A₃R might reduce fat production, improve glucose tolerance, which will not be affected by food intake and may be an good therapy way of typeâ…¡diabetes.Results showed heart rate, body temperature and activity were significantly depressed by 75mg/kg caffeine in mice, while the same dose of other methylxanthines could not mimic the same effect; neither diazepam could antagonize the inhibitory effect of caffeine. Phosphodiesterase (PDE) inhibitors (rolipram and papaverine) could mimic part of the effects of caffeine. The large inhibitory effect of high dose caffeine happened not only in WT mice but also other genotypes mice, except A₃R-/- mice depressed less than WT mice. KitW-sh mast cell-deficient mice were inhibited similarly as A₃R-/- mice by high dose of caffeine. Both A₃R agonist (CI-IB-MECA) and mast cell degranulation compound (C48/80) could induce the fall of body temperature. Histamine level in serum was significantly increased 30min after high dose of caffeine injection, and c-fos mRNA expression in amygdala area was also increased in WT mice 4hr after high dose of caffeine injection, but these were not seen in mice with repeated high dose of caffeine injection. Experiments showed that high dose of caffeine could cause tolerance, and the effect was still existing 2 weeks later. So we conclude here that the depressed effects of high dose of caffeine on heart rate, body temperature and activity in all genotypes mice were different from the stimulant effects of lower dose treament. The depressed effect of high dose of caffeine was not due to working as adenosine antagonist; part of the effect was due to PDE inhibitor and part due to activation of central mechanism (c-fos), through mediating A₃R involved degranulation of mast cells, causing massive release of histamine. Further studies are needed for confirmation.