Puttfarcken P, Werling L L, Brown S R, Cote T E, Cox B M
Mol Pharmacol. 1986 Aug;30(2):81-9.
The effects of varying the sodium concentration (at constant ionic strength) on opioid binding at mu- and delta-opioid receptors in 7315c and NG108-15 cells has been examined. The binding of [3H]etorphine to mu-receptors on 7315c cells was increased by replacing the sodium in the incubation medium with potassium or N-methyl-D-glucamine. This effect was shown to be attributable to an increase in affinity, with no change in the maximum number of binding sites, both in cell membrane suspensions and in intact 7315c cells. Replacement of sodium with potassium or N-methyl-D-glucamine in NG108-15 membrane or intact cell suspensions also resulted in an increase in [3H]etorphine binding, but in these cells the effect was associated with an increase in the number of binding sites measurable under these experimental conditions. The effects of sodium on opioid inhibition of adenylate cyclase in membrane preparations from 7315c and NG108-15 cells also differed. Sodium reduced apparent agonist affinity in 7315c membranes. In NG108-15 cell membranes, sodium was essential for the demonstration of opioid inhibition of cyclase activity. Increasing the sodium concentration above 0.5 mM resulted in an increase in the fraction of total enzyme activity inhibited by opioid, but the opioid IC50 did not change. In the companion paper, it is shown that the effects of sodium removal on mu- and delta-receptor binding in guinea pig brain neural membranes were similar to those observed in the cell preparations. An increase in intracellular sodium concentration without change in extracellular concentration was effected by incubation of 7315c and NG108-15 cells with the sodium-selective ionophore, monensin. When sodium was present in the extracellular medium, monensin reduced [3H]etorphine binding by 50% or more, both at mu-receptors in 7315c cells and at delta-receptors in NG108-15 cells. In the absence of sodium, however, monensin treatment produced only a small inhibition of binding. These results suggest that sodium acts at an intracellular site to regulate opioid agonist binding at both mu- and delta-receptors, but that the mode of regulation is not identical at each site. Since a reduction in intracellular sodium concentration by removal of extracellular sodium increases agonist binding, and an increase in intracellular sodium following monensin treatment reduces agonist binding, it is probable that the intracellular sodium concentration is a critical regulator of opioid agonist binding in intact cells.
研究了在7315c和NG108 - 15细胞中，改变钠浓度（在恒定离子强度下）对μ - 和δ - 阿片受体上阿片类药物结合的影响。用钾或N - 甲基 - D - 葡糖胺替代孵育培养基中的钠，可增加[³H]埃托啡与7315c细胞上μ - 受体的结合。在细胞膜悬液和完整的7315c细胞中，这种效应显示是由于亲和力增加，结合位点的最大数量没有变化。在NG108 - 15细胞膜或完整细胞悬液中用钾或N - 甲基 - D - 葡糖胺替代钠，也导致[³H]埃托啡结合增加，但在这些细胞中，这种效应与在这些实验条件下可测量的结合位点数量增加有关。钠对7315c和NG108 - 15细胞膜制剂中阿片类药物抑制腺苷酸环化酶的作用也不同。钠降低了7315c细胞膜中表观激动剂亲和力。在NG108 - 15细胞膜中，钠对于证明阿片类药物抑制环化酶活性是必需的。将钠浓度增加到0.5 mM以上导致阿片类药物抑制的总酶活性分数增加，但阿片类药物的IC50没有变化。在配套论文中表明，去除钠对豚鼠脑神经元膜中μ - 和δ - 受体结合的影响与在细胞制剂中观察到的相似。用钠选择性离子载体莫能菌素孵育7315c和NG108 - 15细胞，可在细胞外浓度不变的情况下增加细胞内钠浓度。当细胞外培养基中存在钠时，莫能菌素使7315c细胞中的μ - 受体和NG108 - 15细胞中的δ - 受体处的[³H]埃托啡结合减少50%或更多。然而，在没有钠的情况下，莫能菌素处理仅产生对结合的小抑制作用。这些结果表明，钠在细胞内位点起作用，以调节μ - 和δ - 受体上阿片类激动剂的结合，但每个位点的调节模式并不相同。由于去除细胞外钠导致细胞内钠浓度降低会增加激动剂结合，而莫能菌素处理后细胞内钠增加会减少激动剂结合，因此细胞内钠浓度很可能是完整细胞中阿片类激动剂结合的关键调节因子。
