1. As we have shown earlier, pharmacologic inhibition or genetic deletion of A1 adenosine receptors (A1AR) causes marked reductions in tubuloglomerular (TGF) responses, but the location of the A1AR involved in TGF has remained uncertain. To investigate this issue we generated mice with overexpression of A1AR in smooth muscle cells by pronuclear injection of the mouse A1AR cDNA linked to a 5.38 kb fragment of the rat smooth muscle alpha actin containing 2.6 kb of promoter and 2.78 kb of first intron (gift of G.K. Owens, University of Virginia). Two of the 8 founder mice that showed the highest A1AR expression in kidney and heart were selected to establish independent lines of transgenic mice (A1AR-tg). Compared to wild type mice, renal A1AR mRNA expression determined by real time RT-PCR was 353 plus/minus 42 % (n=8) in line 4 and 575 plus/minus 43 % (n=5) in line 7 of A1AR-tg. A1AR mRNA was also elevated in heart, but it was elevated in brain. Ambient urine osmolarity was similar in both A1AR-tg lines (A1AR-tg4: 1678 n=10, and A1AR-tg7: 1668 mosmol/l, n=10), not significantly different from Wt (1294 mosmol/l, n=13). Maximum TGF responses of stop flow pressure (0-30 nl/min flow step) were increased from 8.4 plus/minus 0.9 mm Hg in Wt (n=21) to 14.1 plus/minus 0.7 mm Hg in A1AR-tg4 (n=20; p<.0001), and to 12.3 plus/minus 1.4 mm Hg in A1AR-tg7 (n=10; p<0.02). TGF responses were significantly greater in A1AR-tg than Wt in the intermediate flow ranges of 7.5-10 nl/min and 10-15 nl/min. Basal plasma renin concentration (PRC, ng angI/ml/hr), and by inference renin secretion, was slightly increased in A1AR-tg compared to Wt (A1AR-tg4: 1918 plus/minus 193; A1AR-tg7: 1983 plus/minus 666; Wt: 1251 plus/minus 142; p=.01 and .24 vs. Wt). Suppression of PRC after acute salt loading (5% body weight saline i.v.) was similar in A1AR-tg4 (-36%), A1AR-tg7 (-33%), and Wt (-35%). Thus, macula densa control of renin secretion appeared largely unaltered, possibly reflecting a low activity of the smooth muscle actin promoter in granular JG cells and consequently low levels of transgene expression. We conclude that vascular overexpression of A1AR results in markedly enhanced TGF responsiveness indicating that A1AR expression in vascular smooth muscle cells is critical for TGF signaling. Furthermore, expression levels of A1AR are a determinant of the TGF response magnitude. ? 2. The nephropathy induced by the administration of radiocontrast media accounts for more than 10% of hospital-acquired renal failure. Increased release of renal adenosine and stimulation of renal adenosine receptors have been proposed as major mechanisms in the development of contrast media-induced acute kidney injury. We have therefore assessed the effect of an injection of iodinated radiographic contrast medium on renal blood flow (RBF) and blood pressure (BP) in wild type (WT) mice and mice lacking adenosine A1 receptor (A1AR-/-). In male WT and A1AR-/- mice with a mixed 129J/C57Bl6 genetic background we measured total RBF using a V-type ultrasonic flow probe placed on the renal artery. After achieving stability in the RBF and BP recordings, a dimeric, isosmolar, nonionic, and water-soluble radiographic contrast medium (iodixanol, 320 mg of iodine/ml) was administered intravenously at three doses (25, 50, or 100 microl). Baseline mean BP in WT and A1AR-/- mice was respectively 81 plus/minus 12 mmHg and 83 plus/minus 13 mmHg, and mean RBF was 1.2 plus/minus 0.26 mL/min and 1.2 plus/minus 0.19 mL/min. Intravascular infusion of contrast medium caused a dose dependent increase in BP in both groups of animals. After a short-lasting initial increase of RBF by 50% at the highest dose, CM caused a long-lasting and dose-dependent reduction of RBF that was similar between wild type and A1AR-/- mice both in extent and duration of the effect. We therefore conclude that activation of adenosine 1 receptors do not play a major role in the renal blood flow deficit in A1AR-/- mice after administration of iodinated and isosmolar contrasst media. Given that contrast media have been found to be highly viscous we suspect that the renal blood flow reduction may be a consequence of an increased blood viscosity.? 3. In a recent study it has been reported that intraperitoneal injection of 5-AMP in conscious mice lowers core body temperature (CBT) to below 30 C and induces a state of torpor for an extended period of time (Zhang et al., Nature 2006). We have investigated whether a similar effect is also exerted by adenosine and which A1 adenosine receptors (A1AR) play a role in this response. We found that the A1 agonist cyclopentyladenosine (1 mg/kg) reduced CBT to a nadir of 23.3C at 160 min (n=5) without apparent shivering while 5-AMP induced dose-dependent non-shivering hypothermia and torpor at doses between 55 and 550 mg/kg. At the highest dose, AMP reduced CBT by 10.8 C at 80 min in wild type mice (WT; n=5) while CBT fell by only 6.6 C in A1AR-/- mice (n=5; p<0.001). The time of CBT to return to normal was about 4 hrs in WT and 2 hrs in A1AR-/-mice. The fall in CBT was not associated with an upregulation of uncoupling protein 1 (UCP1) in brown fat whereas a reduction in room temperature striongly stimulated UCP1 expression. As measured by telemetry, the time course of the hypothermic state was paralleled by marked blood pressure and heart rate reductions (to a nadir of 40 mm Hg and 200 bpm in WT, and 60 mm Hg and 400 bpm in A1AR-/-). At 550 mg/kg, locomotor activity was completely suppressed between 45 and 210 min in WT, and between 60 and 120 min in A1AR-/-. In contrast to A1AR-/-, AMP-induced hypothermia was not significantly different from WT in CD73-/- mice (-11.1 C plus/minus 0.6; n=5). We conclude that AMP as well as A1AR agonists induce a dose-dependent non-shivering and long-lasting hypothermia in conscious mice, that hypothermia is accompanied by blood pressure and heart reductions, and immobility, and that all of these effects are smaller in A1AR-/- than WT mice. Thus, AMP-induced hypothermia is partly mediated by A1AR. We believe that this is a central effect in which ATP degradation products act as suppressors of thermogenesis.? 4. The availability of adenosine 1 receptor (A1AR)-deficient mice generated in our laboratory has continued to contribute importantly to the recognition of adenosine functions in various organ systems of the body. One of these effects exerted by A1AR activation is a depression of excitatory nerve transmission in the thalamus of the brain that reduces tremor activity. Thus, A1AR-deficient mice demonstrate involuntary movements and seizures during low stimulation levels. Activation of A1AR play an important role in preventing the excitatory side-effects associated with deep brain stimulation, a therapeutic modality in the treatment of tremor and other movement disorders. In the cardiovascular system A1AR were found to enhance the ischemic tolerance of the heart. A1AR are also involved in the adaptive response to chronic systolic overload of the heart. The hypertrophy of cardiac muscle cells and the interstitial fibrosis resulting from aortic constriction was found to be more pronounced in ecto-5' nucleotidase knockout mice in which extracellular adenosine levels were reduced.
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