Nerme V, Severne Y, Abrahamsson T, Vauquelin G
Mol Pharmacol. 1986 Jul;30(1):1-5.
The beta-adrenergic receptor in membranes from cat, rat, and guinea pig hearts appears to undergo spontaneous coupling to the stimulatory nucleotide-regulatory protein, Ns. This was demonstrated by incubating cardiac membranes from acutely reserpinized animals with the alkylating reagent N-ethylmaleimide (NEM), which is known to "freeze" the beta-receptor Ns-complex. The concentration of norepinephrine in these membrane preparations was less than 0.1 nM. Cat heart membranes were preincubated for 10 min at 30 degrees with NEM (10(-7)-10(-3) M) and subsequently incubated with (-)-[125I]iodopindolol (IPIN) (18 min, 30 degrees). NEM caused a concentration-dependent decrease in specific IPIN binding with a maximal reduction of about 20% at 0.1 mM. This decrease occurred even in the absence of MgCl2 (0.5 mM EDTA added as well). A comparable reduction was also observed in myocardial membranes from rat and guinea pig. This fall reflects a decrease in the number of beta-adrenergic receptor sites, as demonstrated by saturation binding experiments with IPIN. The equilibrium dissociation constant of the radioligand for the remaining receptors was not affected. When increasing concentrations of GTP were included in the preincubation mixture, it resulted in a dose-dependent protection of NEM-induced decrease in IPIN binding. The protection was complete at 0.1 mM GTP. In addition, GTP reversed the NEM effect when added to the incubation mixture 10 min after NEM. The apparent reduction in cardiac beta-adrenergic receptor number by NEM (in the absence of beta-receptor agonist) is compatible with a model in which part of the receptor population is able to undergo spontaneous coupling to Ns.
猫、大鼠和豚鼠心脏膜中的β-肾上腺素能受体似乎会自发地与刺激性核苷酸调节蛋白Ns偶联。这是通过将急性利血平化动物的心脏膜与烷基化试剂N-乙基马来酰亚胺（NEM）一起孵育来证明的，已知该试剂会“冻结”β受体-Ns复合物。这些膜制剂中去甲肾上腺素的浓度低于0.1 nM。将猫心脏膜在30℃下与NEM（10^-7 - 10^-3 M）预孵育10分钟，随后与（-）-[125I]碘吲哚洛尔（IPIN）（30℃，18分钟）一起孵育。NEM导致特异性IPIN结合呈浓度依赖性降低，在0.1 mM时最大降低约20%。即使在没有MgCl2（也添加了0.5 mM EDTA）的情况下，这种降低也会发生。在大鼠和豚鼠的心肌膜中也观察到了类似的降低。这种下降反映了β-肾上腺素能受体位点数量的减少，如用IPIN进行的饱和结合实验所证明的那样。放射性配体与其余受体的平衡解离常数不受影响。当预孵育混合物中加入浓度不断增加的GTP时，它会导致剂量依赖性地保护NEM诱导的IPIN结合减少。在0.1 mM GTP时保护作用完全。此外，在NEM加入10分钟后将GTP添加到孵育混合物中时，GTP会逆转NEM的作用。NEM（在没有β受体激动剂的情况下）使心脏β-肾上腺素能受体数量明显减少，这与一部分受体群体能够自发地与Ns偶联的模型是一致的。
