Puttfarcken P S, Werling L L, Cox B M
Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799.
Mol Pharmacol. 1988 May;33(5):520-7.
The effects of prolonged morphine exposure on the mu opioid receptor in 7315c pituitary tumor cell membranes have been examined. Since a low concentration of naloxone reversed the inhibition of forskolin-stimulated adenylyl cyclase induced by the mu-selective agonist, Tyr-D-Ala-Gly-MePhe-Gly-ol (DAGO), and by high concentrations of [D-Pen2-D-Pen5]enkephalin (DPDPE), we suggest that these cells contain a homogeneous population of mu opioid receptors coupled to adenylyl cyclase via a guanyl nucleotide-binding protein. Studies measuring the ability of [D-Ala2-D-Leu5]enkephalin (DADLE), an opioid agonist, to inhibit adenylyl cyclase in cells that had been exposed to 100 microM morphine for varying periods of time, indicated that the agonist no longer inhibited enzyme activity after 5 hr of morphine exposure. Measurements of 3H-antagonist binding in membranes from cells exposed to morphine demonstrated a decreased receptor density after 24 hr of 100 microM morphine exposure with no change in the antagonist affinity. Computer analysis indicated a 20% decrease in the number of mu receptors labeled after 24 hr of morphine exposure and a 60% decrease after 72 hr of exposure. Computer analysis of agonist competition against 3H-antagonist binding confirmed the existence of one binding site with an affinity intermediate between the high and low apparent affinity states observed in membranes from untreated cells. Addition of 10 microM GTP gamma S did not affect the agonist affinity or receptor density in membranes from morphine-treated cells, suggesting that the receptors were uncoupled from G proteins, as observed in 7315c cell membranes that have been treated with pertussis toxin. Thus chronic morphine treatment induced a rapid loss of opioid mu receptor-mediated inhibition of adenylyl cyclase (desensitization), and a more slowly developing reduction in receptor number. The desensitization was accompanied by a loss of guanyl nucleotide regulation of agonist affinity. These findings are comparable to results reported for the delta opioid receptor and the beta-adrenergic receptor upon prolonged agonist exposure.
研究了长期暴露于吗啡对7315c垂体瘤细胞膜中μ阿片受体的影响。由于低浓度的纳洛酮可逆转由μ选择性激动剂酪氨酰-D-丙氨酰-甘氨酰-甲硫苯丙氨酰-甘氨醇（DAGO）以及高浓度的[D-青霉胺2-D-青霉胺5]脑啡肽（DPDPE）所诱导的对福斯高林刺激的腺苷酸环化酶的抑制作用，我们认为这些细胞含有通过鸟苷酸结合蛋白与腺苷酸环化酶偶联的均一的μ阿片受体群体。对阿片类激动剂[D-丙氨酰2-D-亮氨酰5]脑啡肽（DADLE）抑制已暴露于100μM吗啡不同时间的细胞中腺苷酸环化酶能力的研究表明，在吗啡暴露5小时后，该激动剂不再抑制酶活性。对暴露于吗啡的细胞的膜中3H拮抗剂结合的测量表明，在100μM吗啡暴露24小时后受体密度降低，而拮抗剂亲和力没有变化。计算机分析表明，吗啡暴露24小时后标记的μ受体数量减少20%，暴露72小时后减少60%。对激动剂与3H拮抗剂结合竞争的计算机分析证实存在一个结合位点，其亲和力介于未处理细胞的膜中观察到的高和低表观亲和力状态之间。添加10μM GTPγS对吗啡处理细胞的膜中的激动剂亲和力或受体密度没有影响，这表明受体与G蛋白解偶联，正如在用百日咳毒素处理的7315c细胞膜中所观察到的那样。因此，慢性吗啡处理导致阿片μ受体介导的腺苷酸环化酶抑制作用迅速丧失（脱敏），以及受体数量更缓慢地减少。脱敏伴随着激动剂亲和力的鸟苷酸调节丧失。这些发现与长期暴露于激动剂后δ阿片受体和β肾上腺素能受体的报道结果相当。
