Profound hypothermia after adenosine kinase inhibition in A1AR-deficient mice suggests a receptor-independent effect of intracellular adenosine

Abstract

Administration of the nucleoside adenosine has been shown to induce hypothermia in a number of species, an effect mediated predominantly by the adenosine 1 receptor (A1AR) subtype. The present experiments were performed to explore the possibility that the rise of intracellular adenosine levels expected to accompany adenosine administration may contribute to the hypothermic effect of adenosine independent of A1AR activation. Since phosphorylation of adenosine by adenosine kinase (ADK) is causal in the maintenance of low intracellular adenosine, we have examined the effect of ADK inhibition on core body temperature (CBT). Our data show that inhibition of ADK by A-134974 causes a long-lasting deep hypothermia in wild-type mice. Since there was an about 4-fold increase of adenosine plasma levels, experiments were repeated in A1AR-/- mice. ADK inhibition caused deep hypothermia despite the absence of A1AR, although the effect was significantly reduced compared to WT. Furthermore, the dose-dependent hypothermia caused by adenosine administration in WT mice was found to be reduced, but not abolished in A1AR-/- mice. To assess the possible role of A2AR and A3AR activation in our experimental setting, we compared the effects of the agonists CPA (A1AR), CGS21680 (A2AR), and IB-MECA (A3AR) on CBT. Hypothermia induced by CPA was much greater than that caused by CGS21680 or IB-MECA indicating that A1AR activation is the major receptor-dependent pathway for adenosine-induced hypothermia under our experimental conditions. Induction of deep hypothermia by inhibition of ADK, maintenance of this effect in A1AR-/- mice, and maintenance of adenosine-induced hypothermia in A1AR-deficient mice suggest that a receptor-independent action of adenosine requiring intact function of adenosine kinase contributes importantly to the hypothermia induced by adenosine.

Physiology and effects of nucleosides in mice lacking all four adenosine receptors

“…Adenosine kinase phosphorylates adenosine to AMP, keeping intracellular adenosine levels low, thereby drawing extracellular adenosine into cells down its concentration gradient. It has been suggested that inhibition of adenosine kinase causes hypothermia via AR-independent mechanisms [38]. We confirmed that treatment of wild-type mice with the adenosine kinase inhibitor A134974 [39] caused hypothermia in a dose-dependent manner (Fig 5A–5D).…”

“…In contrast, the QKO mice demonstrate that the acute hypothermia caused by an adenosine kinase inhibitor is completely attributable to ARs. Some of the hypothermia is due to the increased extracellular adenosine activating A 1 AR [38]. While possible explanations for the remaining A134974-induced hypothermia have been proposed, the QKO results dictate that the non–A 1 AR-mediated hypothermia must occur via A 2A AR, A 2B AR, and/or A 3 AR.…”

“…Adenosine was calibrated ( R 2 ≥ 0.999) from 1.0 to 2,500 ng/mL and uridine from 0.25 to 750 μg/mL in the original samples. Literature plasma adenosine levels are 100 to 1,000 nM in human [54] and 145 to 6,100 nM in mouse [38,86,87].…”

The Inside Story of Adenosine

“…In addition, fasting-induced torpor in mice does not require any of these receptors, but the mechanism remains unknown [ 20 ]. Eisner et al [ 24 ] demonstrate that treating mice with Ado or AdoK inhibitors (thus obtaining an increase of Ado) causes a profound hypothermia lasting longer if caused by AdoK inhibitors. The same experiment performed in A1AR −/− mice caused a less pronounced effect that is 40–60% of that observed in wild type mice.…”

“…Furthermore, ADA inhibitors are unable to induce hypothermia and do not affect the hypothermic effect of AdoK inhibitors on both wild type and A1AR −/− mice, thus indicating that the deamination of Ado is not required for the nucleoside to induce hypothermia. The authors conclude that hypothermia induced by Ado in mice is a combined response to a receptor-mediated (mainly A1-mediated) mechanism and to a still-unknown intracellular mechanism in which AdoK plays a fundamental regulatory role [ 24 ].…”