Cheung Y D, Barnett D B, Nahorski S R
Biochem Pharmacol. 1984 Apr 15;33(8):1293-8. doi: 10.1016/0006-2952(84)90183-7.
The specific binding of (3H)yohimbine to alpha 2 adrenoceptors in rat cerebral cortex varied significantly with methods of membrane preparation. Membranes prepared in a sucrose containing buffer showed markedly lower Bmax values than those prepared in hypotonic buffer without any change in affinity (Kd) for (3H)yohimbine. Higher concentrations of residual endogenous norepinephrine were found in sucrose prepared membranes. In the presence of exogenous norepinephrine (10(-8), 10(-7) M), membranes prepared in hypotonic buffer showed apparent reduced receptor densities similar to those observed in sucrose prepared cerebral membranes. The presence of exogenous norepinephrine did not produce any apparent change in the overall Kd for (3H)yohimbine. The depression of (3H)yohimbine binding capacities could, in all cases, be reversed by performing incubations in the presence of 200 mM NaCl and 10 microM Gpp(NH)p which synergistically dramatically reduce the affinity of norepinephrine for cerebral alpha 2 adrenoceptors. It is concluded, therefore, that the lower (3H)yohimbine binding capacities in sucrose prepared membranes appears to be due to the occupancy of receptor sites by residual norepinephrine in a pseudo non competitive manner. The reduced affinity of retained endogenous norepinephrine for the alpha 2 adrenoceptor, in the presence of Na+ and guanine nucleotides, would seem to be the cause of the apparent increase in (3H)yohimbine binding sites produced by these modulators in sucrose prepared membranes.
（3H）育亨宾与大鼠大脑皮层α2肾上腺素能受体的特异性结合随膜制备方法的不同而有显著差异。在含蔗糖缓冲液中制备的膜显示出的Bmax值明显低于在低渗缓冲液中制备的膜，而对（3H）育亨宾的亲和力（Kd）没有变化。在蔗糖制备的膜中发现了更高浓度的残留内源性去甲肾上腺素。在外源性去甲肾上腺素（10^(-8)，10^(-7) M）存在的情况下，在低渗缓冲液中制备的膜显示出明显降低的受体密度，类似于在蔗糖制备的大脑膜中观察到的情况。外源性去甲肾上腺素的存在对（3H）育亨宾的总体Kd没有产生任何明显变化。在所有情况下，（3H）育亨宾结合能力的降低都可以通过在200 mM NaCl和10 μM Gpp(NH)p存在下进行孵育来逆转，这两种物质协同作用可显著降低去甲肾上腺素对大脑α2肾上腺素能受体的亲和力。因此可以得出结论，在蔗糖制备的膜中较低的（3H）育亨宾结合能力似乎是由于残留去甲肾上腺素以假非竞争性方式占据受体位点所致。在Na+和鸟嘌呤核苷酸存在的情况下，保留的内源性去甲肾上腺素对α2肾上腺素能受体亲和力的降低，似乎是这些调节剂在蔗糖制备的膜中使（3H）育亨宾结合位点明显增加的原因。
