Gasic S, Green A
Department of Internal Medicine, University of Texas Medical Branch, Galveston 77555-1060.
Biochem Pharmacol. 1995 Mar 15;49(6):785-90. doi: 10.1016/0006-2952(94)00537-v.
Prolonged treatment of rat adipocytes with the A1-adenosine receptor agonist [-]N(6)-phenylisopropyl adenosine (PIA) or prostaglandin E1 down-regulates Gi and induces heterologous desensitization. alpha 2-Adrenergic receptors also inhibit adenylyl cyclase through Gi, but whether alpha 2-receptors are present on rat adipocytes has been controversial. We have investigated the effects of the highly specific alpha 2-adrenergic agonist UK 14304 (5-bromo-6-[2-imidazolin-2-ylamino]-quinoxaline) on rat adipocytes. In previous studies on young rats, we were unable to demonstrate an effect of the alpha 2-agonist. We now report that, in cells isolated from older, more obese rats (300-400 g), UK 14304 inhibited lipolysis (measured as the rate of glycerol release) by approximately 40% (EC50 approximately 40 nM). To determine whether UK 14304 would induce heterologous desensitization, we incubated adipocytes with or without 1 microM UK 14304 for 4 days in primary culture. The cells were then washed, and the rate of lipolysis was determined during a 30-min incubation in the presence of various concentrations of PIA. The concentration-response curve for PIA-induced inhibition of lipolysis was shifted to the right, with the EC50 for UK 14304-treated cells about 2-fold higher than in the control cells. This finding demonstrates that the alpha 2-agonist can desensitize the response to PIA and indicates heterologous desensitization. To investigate the mechanism of this phenomenon, we isolated crude membrane fractions from the cells and analyzed them on Western blots using antibodies against Gi alpha 1, 2 and 3. In cells treated with UK 14304 for 4 days, Gi1 alpha and Gi2 alpha were down-regulated to about 15% of the control level, and Gi3 alpha was decreased to 30% of control. We conclude that prolonged treatment of adipocytes with the alpha 2-agonist induces heterologous desensitization of lipolysis and causes down-regulation of Gi. The findings suggest that G-protein down-regulation is a mechanism for heterologous desensitization.
用A1 - 腺苷受体激动剂[-]N(6)-苯基异丙基腺苷（PIA）或前列腺素E1对大鼠脂肪细胞进行长时间处理会下调Gi并诱导异源脱敏。α2 - 肾上腺素能受体也通过Gi抑制腺苷酸环化酶，但大鼠脂肪细胞上是否存在α2 - 受体一直存在争议。我们研究了高特异性α2 - 肾上腺素能激动剂UK 14304（5 - 溴 - 6 - [2 - 咪唑啉 - 2 - 基氨基] - 喹喔啉）对大鼠脂肪细胞的影响。在之前对幼鼠的研究中，我们未能证明α2 - 激动剂的作用。我们现在报告，在从年龄较大、更肥胖的大鼠（300 - 400克）分离的细胞中，UK 14304抑制脂解作用（以甘油释放速率衡量）约40%（半数有效浓度约为40 nM）。为了确定UK 14304是否会诱导异源脱敏，我们在原代培养中用或不用1 μM UK 14304孵育脂肪细胞4天。然后洗涤细胞，并在存在不同浓度PIA的情况下孵育30分钟期间测定脂解速率。PIA诱导的脂解抑制浓度 - 反应曲线向右移动，UK 14304处理细胞的半数有效浓度比对照细胞高约2倍。这一发现表明α2 - 激动剂可使对PIA的反应脱敏，并表明存在异源脱敏。为了研究这种现象的机制，我们从细胞中分离出粗膜组分，并使用针对Giα1、2和3的抗体在蛋白质免疫印迹上进行分析。在用UK 14304处理4天的细胞中，Gi1α和Gi2α下调至对照水平的约15%，Gi3α降至对照的30%。我们得出结论，用α2 - 激动剂对脂肪细胞进行长时间处理会诱导脂解的异源脱敏并导致Gi下调。这些发现表明G蛋白下调是异源脱敏的一种机制。
