TONIC ACTIVITY OF ADENOSINE A1-ADENOSINE RECEPTORS

多年来，人们已经认识到腺苷是白色脂肪组织中脂解、腺苷和腺苷环化酶活性的有力和有效的抑制剂。白色脂肪组织中的A1-腺苷受体在张力上是活跃的，用脂肪细胞中A1-腺苷受体拮抗剂治疗是自发活跃的，即使在没有激动剂的情况下也会导致抑制脂肪分解，(2)腺苷的抗脂作用有很高的受体储备，因此激活一小部分受体就足以引起功能反应，(3)可能存在一个细胞内A1-腺苷受体池，其腺苷浓度超过细胞外空间的浓度。我们实验室最近的研究表明，在转基因的中国仓鼠卵巢细胞和大鼠附睾脂肪组织制备的膜上，都存在A1-腺苷受体的非激动剂依赖性活性。脂肪细胞有高密度的A1-腺苷受体，这一发现与自发受体活性的存在和高受体储备相一致。脂肪细胞内有大量的膜泡。文献报道提示小窝可能是腺苷受体信号转导的部位。因此，研究的具体目的是：(1)确定在没有激动剂的情况下，完整脂肪细胞中的A1-腺苷受体是否活跃；(2)确定腺苷的受体储备，以抑制脂肪细胞中环磷酸腺苷的积聚和减少脂肪细胞中的脂肪分解；(3)检验A1-腺苷受体池介导腺苷的紧张性作用以抑制cAMP积聚的假设。大鼠分离的附睾脂肪细胞将用于这些研究。A1-腺苷受体的非激动剂非激动剂活性将在Aim 1中使用反向激动剂和中性拮抗剂进行研究，这些激动剂和中性拮抗剂已经在我们实验室中得到了表征。在AIM 2中，将使用FSCPX来测量腺苷的受体储备，FSCPX是我们实验室鉴定的A1-腺苷受体的不可逆拮抗剂。Aim 3将使用最近描述的测定受体室内腺苷浓度和分离空泡膜进行研究的方法。这些研究结果将解释脂肪细胞A1-腺苷受体紧张性活动的药理学基础。这项研究可以作为研究A1-腺苷受体与肥胖相关的过度活性的前奏，可以用来研究调节自发A1-腺苷受体活性的事件，以及脂肪细胞中腺苷产生和信号的亚细胞位置(包括激动剂依赖和非激动剂非依赖)。

For many years it has been recognized that adenosine is a potent and efficacious inhibitor of lipolysis and adenylate and adenylate cyclase activity in white adipose tissue. A1-adenosine receptors in white adipose tissue are tonically active and treatment with antagonists of the A1- adenosine receptors in adipocytes are spontaneously active and cause inhibition of lipolysis even in the absence of an agonist, (2) there is a high receptor reserve for the anti-lipolytic action of adenosine, such that activation of a small fraction of receptors is sufficient to cause a functional response, and (3) there may exist an intracellular pool of A1- adenosine receptors in equilibrium with a concentration of adenosine which exceeds that in the extracellular space. Recent studies in our laboratory have demonstrated the presence of agonist-independent activity of A1-adenosine receptors both in transfected Chinese hamster ovary cells and in membranes prepared from rat epididymal adipose tissue. Adipocytes have a high density of A1-adenosine receptors, a finding consistent with both the presence of spontaneous receptor activity and a high receptor reserve. Membrane caveolae are numerous in adipocytes. Literature reports suggest that caveolae may be a site of adenosine receptor signaling. Therefore the specific aims of the research are (1) determine whether A1-adenosine receptors in the intact adipocyte are active in the absence of an agonist, (2) determine the receptor reserves for adenosine to inhibit accumulation of cyclic AMP and to reduce lipolysis in adipocytes, (3) test the hypothesis that an "intracellular" pool of A1-adenosine receptors mediates a tonic effect of adenosine to inhibit the accumulation of cAMP. Rat isolated epididymal adipocytes will be used for these studies. Agonist independent activity of A1-adenosine receptors will be investigated in Aim 1 by use of inverse agonists and neutral antagonists that have been previously characterized in our laboratory. Receptor reserve for adenosine will be measured in Aim 2 by use of FSCPX, an irreversible antagonist of the A1-adenosine receptor that has been characterized in our laboratory. Recently described methods to determine the concentration of adenosine in the receptor compartment and to isolate caveolar membranes for study will be used in Aim 3. The results of the studies will explain the pharmacologic basis of tonic activity of adipocyte A1-adenosine receptors. The research can be used a prelude to investigation of the excessive activity of A1-adenosine receptors associated with obesity, to investigation of events that regulate spontaneous A1-adenosine receptor activity, and to investigation of subcellular sites of adenosine production and signaling (both agonist- dependent and agonist-independent) in adipocytes.