Adenosine in Renal Sympathetic Neurotransmission

描述（由申请方提供）：在研究BG 9928（一种腺苷A1型受体的选择性拮抗剂，正在开发作为心力衰竭患者的利尿剂）的肾脏效应时，我们观察到BG 9928在体内可显著降低肾交感神经刺激（RSNS）诱导的肾血管收缩。这一发现表明，肾神经效应器接头的A1受体增强肾交感神经传递。尽管这一假设与我们发表的RSNS增加腺苷释放的报告和其他人发表的交感神经刺激释放腺苷前体ATP以及从ATP形成腺苷的酶的报告一致，但其他人的许多研究清楚地证实，连接前A1受体抑制而不是增加NE释放。总之，这些观察结果表明，如果A1受体参与增强肾交感神经传递，效果必须是后连接。由于A1受体众所周知可增强血管紧张素II诱导的肾血管收缩，我们推测RSNS可增加神经效应器接头中的腺苷和NE，并且腺苷通过接头后A1受体增强NE诱导的血管收缩（通过同步信号传导，即，信号传导的会聚），导致RSNS诱导的肾血管收缩的A1受体增强。因此，本项目的总体目标是确定肾神经效应器接头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研究这一假设：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）; 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中测定（野生型和A1受体敲除）肾，在存在和不存在选择性A1受体拮抗剂的情况下，高度选择性A1受体激动剂对去甲肾上腺素诱导的肾血管收缩的作用; 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重建是否会增强肾血管对RSNS的反应; 5）我们将在野生型小鼠的肾脏中确定肾血管平滑肌细胞中A1受体的基于慢病毒的shRNA选择性敲低是否抑制对RSNS的肾血管反应。这个项目将确定一个主要的机制，有助于肾交感神经传递。由于肾交感神经系统参与了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生理学家在肾功能和功能障碍的各个方面的工作，并将提供一个强有力的理由，使用A1受体阻滞剂作为利尿剂与肾交感神经张力增加的条件。

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.