Adenosine in Renal Sympathetic Neurotransmission

描述(申请人提供)：在研究BG9928(一种选择性的A1型腺苷受体拮抗剂，正被开发为心力衰竭患者的利尿剂)的肾脏效应时，我们在体内观察到BG9928显著减少了肾交感神经刺激(RSNs)引起的肾血管收缩。这一发现向我们表明，肾神经效应器连接处的A1受体增强了肾交感神经传递。尽管这一假说与我们发表的RSNs增加腺苷释放的报道以及其他人发表的关于交感神经刺激释放腺苷前体ATP以及由ATP形成腺苷的酶的报告是一致的，但其他人的大量研究清楚地证明，突触前A1受体抑制而不是增强NE的释放。综上所述，这些观察表明，如果A1受体参与增强肾交感神经传递，其作用肯定是在连接后。由于A1受体可以增强血管紧张素II诱导的肾血管收缩，我们假设RSNs增加神经效应连接处的腺苷和NE，并且腺苷通过连接后的A1受体增强NE诱导的血管收缩(通过一致信号，即信号汇聚)，导致A1受体增强RSNs诱导的肾血管收缩。因此，这个项目的总体目标是确定肾神经效应器连接中的内源性腺苷是否通过A1腺苷受体显著增强对肾交感神经刺激的肾血管收缩反应。我们将使用多种方法来研究这一假说：1)我们将在大鼠和小鼠肾脏中确定选择性A1受体拮抗剂对RSNs诱导的肾血管收缩和嘌呤释放的影响(通过质谱仪)；2)我们将在小鼠肾脏中确定A1受体敲除对RSNs诱导的肾血管收缩和嘌呤释放的影响；3)我们将在大鼠和小鼠(野生型和A1受体敲除)肾脏中确定高选择性A1受体激动剂在有或没有选择性A1受体拮抗剂的情况下对去甲肾上腺素诱导的肾血管收缩的影响；4)我们将在A1受体敲除小鼠的小鼠肾脏中确定基于慢病毒的A1受体选择性在肾血管平滑肌细胞中的重建是否增强了对RSNs的肾血管反应；5)我们将在野生型小鼠的小鼠肾脏中确定基于慢病毒的A1受体选择性shRNA敲除是否抑制了对RSNs的肾血管反应。该项目将确定促进肾交感神经传递的主要机制。由于肾交感神经系统参与大多数肾脏生理和病理生理过程，这一发现将启发和告知肾脏生理学家在肾功能和功能障碍的各个方面工作，并将为在与肾交感神经张力增加相关的情况下使用A1受体阻滞剂作为利尿剂提供强有力的理论依据。

DESCRIPTION (provided by applicant): While investigating the renal effects of BG9928 (a selective antagonist of adenosine type A1 receptors that is being developed as a diuretic for heart failure patients) we observed that in vivo BG9928 profoundly reduces renal vasoconstriction induced by renal sympathetic nerve stimulation (RSNS). This finding suggests to us that A1 receptors in the renal neuroeffector junction augment renal sympathetic neurotransmission. Although this hypothesis is consistent with our published reports that RSNS increases adenosine release and published reports by others that sympathetic nerve stimulation releases the adenosine precursor ATP as well as enzymes that form adenosine from ATP, numerous studies by others clearly establish that prejunctional A1 receptors inhibit, not augment, NE release. Taken together, these observations suggest that if A1 receptors participate in enhancing renal sympathetic neurotransmission, the effect must be post-junctional. Because A1 receptors are well known to enhance angiotensin II-induced renal vasoconstriction, we postulate that RSNS increases adenosine and NE in the neuroeffector junction, and that adenosine, via the post-junctional A1 receptor, enhances NE-induced vasoconstriction (through coincident signaling, i.e., convergence of signaling) resulting in A1 receptor-intensification of RSNS-induced renal vasoconstriction. Accordingly, the overall objective of this project is to determine whether endogenous adenosine in the renal neuroeffector junction and via the A1 adenosine receptor significantly intensifies the renal vasoconstrictor response to renal sympathetic nerve stimulation. We will investigate this hypothesis using multiple approaches: 1) We will determine in rat and mouse kidneys the effects of selective A1 receptor antagonists on RSNS-induced renal vasoconstriction and purine release (by mass spectrometry); 2) We will determine in mouse kidneys the effects of A1 receptor knockout on RSNS-induced renal vasoconstriction and purine release; 3) We will determine in rat and mouse (both wild-type and A1 receptor knockout) kidneys the effects of a highly selective A1 receptor agonist on norepinephrine-induced renal vasoconstriction in the presence and absence of selective A1 receptor antagonists; 4) We will determine in mouse kidneys from A1 receptor knockout mice whether lentiviral-based reconstitution of the A1 receptor selectively in renal vascular smooth muscle cells augments renovascular responses to RSNS; and 5) We will determine in mouse kidneys from wild-type mice whether lentiviral-based shRNA knockdown of the A1 receptor selectively in renal vascular smooth muscle cells inhibits renovascular responses to RSNS. This project will identify a MAJOR mechanism that contributes to renal sympathetic neurotransmission. Because the renal sympathetic nervous system is involved in most renal physiological and pathophysiological processes, this discover would enlighten and inform renal physiologists working in all aspects of renal function and dysfunction and would provide a strong rationale for using A1 receptor blockers as diuretics in conditions associated with increased renal sympathetic tone.