Renal and extrarenal effects of adenosine 1 receptors

腺苷 1 受体的肾内和肾外作用

• 批准号: 7593610
• 负责人: Jurgen Schnermann
• 金额: $ 36.5万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7593610
• 关键词: Adenosine; Adenosine A1 Receptor; Affect; Agreement; Animals; Anticonvulsants; Attenuated; Blood Pressure; Body Fluids; Brain; Breeding; Caffeine; Calcium; Calcium Channel; Cardiac; Cardiovascular system; Chloride Channels; Circadian Rhythms; Classification; Coffee; Complication; Condition; Constriction procedure; Consumption; Cyclic AMP; DNA; Data; Event; Fluorescence; Fura-2; Gene Duplication; Genes; Genetic; Glioblastoma; Glomerular Capillary; Glomerular Filtration Rate; Growth; Heart; Heart Rate; Homeostasis; Hour; IAA-94; Individual; Inflammation; Injury; Intake; Ischemia; Kidney; Knockout Mice; Ligands; Link; Liquid substance; Measurement; Measures; Mediating; Metabolic; Microglia; Motor Activity; Mouse Strains; Mus; Mutation; Neuraxis; Nifedipine; Nucleosides; Nucleotides; Numbers; Organ; Organism; Pathway interactions; Perfusion; Peripheral; Phase; Phenotype; Phospholipase C; Production; Protein Dephosphorylation; Purinergic P1 Receptors; Range; Rate; Receptor Gene; Regulation; Renal Blood Flow; Renal function; Renin; Resistance; Reticulum; Role; Signal Pathway; Smooth Muscle Myocytes; Surface; Technology; Testing; Thapsigargin; Therapeutic; Translating; Translation Process; Traumatic Brain Injury; Tubular formation; Variant; Vascular resistance; Vasoconstrictor Agents; Wild Type Mouse; adenosine receptor activation; arteriole; body system; calphostin C; capillary bed; cytotoxicity; falls; genetic strain; inhibitor/antagonist; inositol-1,4,5-triphosphate receptor; kidney vascular structure; knockout animal; knockout gene; mutant; null mutation; patch clamp; pressure; prevent; receptor; receptor coupling; receptor expression; research study; response; tool; transmission process; vasoconstriction

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).

1。几年前，我们生成了A1腺苷受体（A1AR）缺陷小鼠的第一行。这些小鼠的遗传背景是129J和C57BL/6 DNA之间的混合物。尽管这些小鼠很有用，但很明显，遗传背景必须均匀，以允许特定基因缺陷及其野生型表型之间的背景无关比较。在A1AR的特殊情况下，特定并发症是由于A1AR基因座与肾素基因座的近端相近，因此这两个基因与遗传联系在一起。这是一种小鼠特异性的现象，某些小鼠菌株具有正常的单肾素基因，而其他小鼠菌株由于基因重复事件而具有两个肾素基因。由于诱导无效突变的129J DNA具有两个肾素基因，因此遗传129J/C57BL/6动物中的基因敲除小鼠始终有两个肾素基因，而野生型对照只有一个。因此，在这种特殊情况下，我们知道野生型和突变动物之间存在系统的差异。因此，我们将A1AR缺失繁殖为两种不同的纯遗传菌株，即C57BL/6和黑色瑞士菌株。后一种菌株具有两个肾素基因，因此在这种情况下，野生型和无效突变体具有相同的肾素基因星座。通过将放射性递质技术应用于血压测量，我们检查了具有不同遗传背景的A1AR缺陷小鼠的24小时血压变化。 虽然A1AR缺乏不影响血压和心率的昼夜节律变化，但我们观察到，原始C57BL/6和黑瑞士背景的A1AR缺陷小鼠在活性阶段的平均动脉血压升高，而与其各自的野生型对照相比，在C57BL/6 Backents型中相同。此外，在混合和C57BL/6背景的A1AR缺陷小鼠中显着增强了自发的运动活性，就像人们对咖啡因样作用所期望的那样，但是在黑瑞士背景中，这种效果不太明显。因此，目前未知的修饰基因基因会影响A1AR缺乏症的表型，这一观察结果可能与觉醒对咖啡消费的觉醒反应中的个体变异性有关。 2。肾脏肾小球损伤通常是由于肾小球毛细血管床暴露于长期升高的血压的结果。通常，在称为自动调节的现象中，肾小球动脉抗性的压力依赖性调节通常会阻止外周动脉压向肾小球毛细血管簇。 负责自动调节的一种机制依赖于由于压力扰动而导致的NaCl浓度的管状变化，并转化为小动脉抗性的变化。 A1AR已被证明对于此翻译过程至关重要，现在我们已经证明它们也重要地参与了自动调节。使用删除A1腺苷受体（A1AR）基因的小鼠，我们已经测试了以下预测：先前由于A1AR缺乏症而建立的TGF与自动调节效率的降低有关。在麻醉的野生型和A1AR缺陷型小鼠中，我们确定了通过上下主动脉瘤降低肾脏灌注压力之前和之后，我们确定了肾小球滤过率（GFR）和肾血流（RBF）。响应于血压降低约15 mM的GFR和RBF在A1AR缺陷方面的下降明显高于野生型小鼠。与A1AR+/+小鼠相比，A1AR - / - 的GFR和RBF（每次变化的GFR或RBF变化之比）的自动调节犹豫不决明显更高。这些结果表明，在A1AR缺陷型小鼠中，肾血管耐药性的自动调节较少，这可能与这些动物中没有TGF调节有关的作用。在维持肾小球完整性需要肾小球毛细管压力的恒定范围内，A1AR有助于防止肾脏损伤。 3。在先前从小鼠肾脏的分离和灌注传入的小动脉的研究中，我们表明腺苷通过磷脂酶C的GI依赖性激活引起血管收缩。现在，我们已经进一步探索了腺苷引起小动脉血管促进的信号传导途径。我们已经观察到腺苷显着增加了通过Fura-2荧光测量的小鼠分离的传入小动脉中细胞内钙浓度。用Thapsigargin（2微摩尔）进行预处理，阻止了腺苷的血管收缩作用，表明从IP3刺激的肌胞浆网（SR）释放钙参与了传统动脉的腺苷收缩机制。与该概念一致的是，IP3受体的抑制剂2-APB（100微摩尔）阻断了腺苷诱导的收缩，而PKC抑制剂Calphostin C没有影响。钙活化的氯化物通道抑制剂IAA-94（30微摩尔）抑制了腺苷介导的收缩。斑块夹的实验表明，腺苷处理诱导了胶质性平滑肌细胞中的去极化电流，该电流被IAA-94废除。此外，腺苷引起的血管收缩受到5个微摩尔硝基胺的显着抑制，表明依赖电压依赖性钙通道参与。我们的数据表明，腺苷通过增加SR释放钙导致的细胞内钙浓度来介导传入小动脉的血管收缩，然后激活Ca2+激活的氯化物通道，从而导致钙化和钙通过电压依赖钙通道引起钙的去变异和钙的涌入。 4。在一项广泛的合作努力中，我们研究了腺苷1受体（A1AR）在人体各种器官系统中的作用。腺苷的各种作用强烈建议，包括对血压的影响，包括对血压的影响，腺苷受体拮抗剂咖啡因施加。在没有A1AR的情况下，液体摄入量升高，对咖啡因不反应，这表明A1AR激活降低了液体的摄入量（6）。在心血管系统中，发现A1AR可增强心脏的缺血性耐受性（5,8,9）。在中枢神经系统中，A1AR缺乏引起了实验性创伤性脑损伤的急剧恶化，揭示了A1AR在脑创伤中的重要抗惊厥作用（4）。此外，在小胶质细胞中表达的A1AR减弱了实验性胶质母细胞瘤的生长（7）。