1. Several years ago we have generated the first line of A1 adenosine receptor (A1AR)-deficient mice. The genetic background of these mice was a mix between 129J and C57Bl/6 DNA. While these mice have been useful, it is abundantly clear that the genetic background has to be uniform to permit background-independent comparisons between specific gene deficiencies and their wild type phenotypes. In the particular case of A1AR a specific complication arises from the fact that the A1AR locus is in close proximity to the renin locus so that these two genes are genetically linked. It is a mouse-specific phenomenon that some mouse strains have the normal single renin gene, while other mouse strains have two renin genes due to a gene duplication event. Because the 129J DNA, the DNA in which the null mutation was induced, has two renin genes, knockout mice in the genetic 129J/C57Bl/6 animals will always have two renin genes while wild type controls will have only one. Thus, in this particular case we are aware of a systematic difference between wild type and mutant animals. We have therefore bred the A1AR deletion into two different pure genetic strains, the C57Bl/6 and Black Swiss strains. The latter strain has two renin genes so that in this case both wild type and null mutants have the same renin gene constellation. By applying radiotransmitter technology for blood pressure measurement we have examined the 24 hour blood pressure variations in A1AR-deficient mice with different genetic backgrounds. While the A1AR deficiency does not affect the circadian variation of blood pressure and heart rate, we have observed that A1AR-deficient mice on the original C57Bl/6 and Black Swiss background have an elevated mean arterial blood pressure in the active phase compared to their respective wild type controls whereas the same mutation on a C57Bl/6 background does not cause this phenotype. Furthermore, spontaneous locomotor activity was markedly enhanced in A1AR-deficient mice on a mixed and C57Bl/6 background as one might expect from a caffeine-like action, but this effect was less pronounced in the Black Swiss background. Thus, currently unknown modifier genes affect the phenotype of A1AR deficiency, an observation that may relate to the individual variability in the awakening response to coffee consumption.? 2. Renal glomerular injury is often the result of exposure of the glomerular capillary bed to an chronically elevated blood pressure. Transmission of peripheral arterial pressure into the glomerular capillary tuft is normally prevented by pressure-dependent adjustments of glomerular arteriolar resistance in a phenomenon called autoregulation. One mechanism responsible for autoregulation relies on sensing tubular changes in NaCl concentration that result from the pressure perturbation and that are translated into changes of arteriolar resistance. A1AR have been shown to be critical for this translation process, and we have now shown that they are also importantly involved in autoregulation. Using mice with deletion of the A1 adenosine receptor (A1AR) gene we have tested the prediction that the absence of TGF, previously established to result from A1AR deficiency, is associated with a reduction in the efficiency of autoregulation. In anesthetized wild type and A1AR-deficient mice we determined glomerular filtration rate (GFR) and renal blood flow (RBF) before and after reducing renal perfusion pressure through a suprarenal aortic clamp. In response to a blood pressure reduction by about 15 mm both GFR and RBF fell significantly more in A1AR-deficient than wild type mice. Autoregulatory indeces for both GFR and RBF (the ratio of the GFR or RBF change per change in perfusion pressure) were significantly higher in A1AR-/- compared to A1AR+/+ mice indicating reduced regulatory responsiveness in the knockout animals. These results suggest that autoregulation of renal vascular resistance is less complete in A1AR-deficient mice, an effect that is presumably related to absence of TGF regulation in these animals. To the extent that constancy of glomerular capillary pressure is required for maintaining glomerular integrity, A1AR contribute to protection against renal injury.? 3. In previous studies in isolated and perfused afferent arterioles from the mouse kidney we have shown that adenosine causes vasoconstriction by Gi-dependent activation of phospholipase C. We have now further explored the signaling pathways by which adenosine causes arteriolar vasoconstriction. We have observaed that adenosine significantly increased the intracellular calcium concentration in mouse isolated afferent arterioles measured by fura-2 fluorescence. Pre-treatment with thapsigargin (2 micromolar) blocked the vasoconstrictor action of adenosine indicating that release of calcium from the sarcoplasmatic reticulum (SR), stimulated presumably by IP3, is involved in the adenosine contraction mechanism of the afferent arteriole. In agreement with this notion is the observation that 2-APB (100 micromolar), an inhibitor of IP3 receptors, blocked the adenosine-induced constriction whereas the PKC inhibitor calphostin C had no effect. The calcium-activated chloride channel inhibitor IAA-94 (30 micromolar) inhibited the adenosine mediated constriction. Patch clamp experiments showed that adenosine treatment induced a depolarizing current in preglomerular smooth muscle cells which was abolished by IAA-94. Furthermore, the vasoconstriction caused by adenosine was significantly inhibited by 5 micromolar nifedipine suggesting involvement of voltage dependent calcium channels. Our data suggest that adenosine mediates vasoconstriction of afferent arterioles through an increase in intracellular calcium concentration resulting from release of calcium from the SR followed by activation of Ca2+-activated chloride channels that causes depolarization and influx of calcium through voltage-dependent calcium channels.? 4. In an extensive collaborative effort we have studied the role of adenosine 1 receptors (A1AR) in various organ systems of the body. Organism-wide actions of adenosine are strongly suggested by the multiplicity of effects, including effects on blood pressure, exerted by the adenosine receptor antagonist caffeine. Fluid intake was elevated in the absence of A1AR and non-responsive to caffeine suggesting that A1AR activation reduces fluid intake (6). In the cardiovascular system A1AR were found to enhance the ischemic tolerance of the heart (5,8,9). In the central nervous system, A1AR deficiency caused a dramatic worsening of an experimental traumatic brain injury revealing an important anticonvulsant action of A1AR in brain trauma (4). Furthermore, A1AR expressed in microglial cells attenuate the growth of experimental glioblastoma (7).
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