TONIC ACTIVITY OF ADENOSINE A1-ADENOSINE RECEPTORS

多年来，已经认识到腺苷是一种强有力的和有效的抑制剂脂解和腺苷酸和腺苷酸环化酶活性在白色脂肪组织。白色脂肪组织中的A1-腺苷受体具有强直活性，即使在没有激动剂的情况下，脂肪细胞中的A1-腺苷受体拮抗剂也会自发激活并导致脂肪分解的抑制。(2)腺苷的抗脂解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性反应。(3)细胞内可能存在一个平衡的A1-腺苷受体池，其腺苷浓度超过细胞外空间。我们实验室最近的研究表明，a1 -腺苷受体在转染的中国仓鼠卵巢细胞和大鼠附睾脂肪组织制备的膜中都存在不依赖激动剂的活性。脂肪细胞具有高密度的a1 -腺苷受体，这一发现与自发受体活性和高受体储备的存在一致。脂肪细胞中膜小泡大量存在。文献报道表明，小泡可能是腺苷受体信号传导的一个位点。因此，本研究的具体目的是：(1)确定完整脂肪细胞中a1 -腺苷受体在缺乏激动剂的情况下是否具有活性；(2)确定腺苷受体的储备，以抑制环状AMP的积累，减少脂肪细胞中的脂解；(3)验证“细胞内”a1 -腺苷受体池介导腺苷的补品作用，抑制cAMP的积累。大鼠离体附睾脂肪细胞将用于这些研究。a1腺苷受体的激动剂独立活性将在Aim 1中通过使用逆激动剂和中性拮抗剂进行研究，这些拮抗剂已经在我们的实验室中进行了表征。腺苷受体储备将在Aim 2中通过使用FSCPX来测量，FSCPX是我们实验室鉴定的a1 -腺苷受体的不可逆拮抗剂。最近描述的测定受体室中腺苷浓度和分离腔泡膜的方法将用于Aim 3。本研究结果将解释脂肪细胞a1 -腺苷受体滋补活性的药理学基础。该研究可用于研究与肥胖相关的a1 -腺苷受体的过度活性，研究调节自发a1 -腺苷受体活性的事件，以及研究脂肪细胞中腺苷产生和信号传导（激动剂依赖性和非激动剂依赖性）的亚细胞位点。

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.