Refahi-Lyamani F, Saadouni S, Costentin J, Bonnet J J
EP 076 du C.N.R.S., U.F.R. de Médecine and Pharmacie de Rouen, Saint Etienne du Rouvray, France.
Naunyn Schmiedebergs Arch Pharmacol. 1995 Feb;351(2):136-45. doi: 10.1007/BF00169327.
We have compared the effect of treating rat striatal cell membranes with ionic hydrophilic sulfhydryl reagents on the specific bindings of [3H]cocaine and of [3H]GBR 12783 (1-[2-(diphenylmethoxy)ethyl]4-(3-phenyl-2-[1-3H]propenyl)-piperaz ine) to the neuronal transporter of dopamine. Treatment with 1 mmol/l 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) resulted in similar time- and concentration-dependent reductions of the specific binding of both radioligands. None of the uptake blockers tested afforded any protection against 1 mmol/l DTNB. Addition of (sub)millimolar concentrations of CaCl2 or MgCl2, or 250 mmol/l KCl to a treatment medium containing 10 mmol/l Na+ significantly increased the DTNB-induced reduction of the specific binding of both radioligands. Cations were likely to be responsible for this effect since ions in combination with DTNB induced similar reductions in binding when either 1 mmol/l CaCl2 or 50-250 mmol/NaCl were added. Effects of cations on the DTNB-induced inhibition of binding were generally more marked on [3H]GBR 12783 than on [3H]cocaine binding. When added to a medium containing 10 mmol/l Na+ 1 mmol/l DTNB induced a reduction in the Bmax of the specific binding of both radioligands. Addition of 1 mmol/l Ca2+ maintained or increased this Bmax reduction and elicited a decrease in affinity which was significant for [3H]GBR 12783 binding. Treatment of membranes with the sodium salt of p-hydroxymercurybenzenesulfonate (pHMBS) induced time- and concentration-dependent decreases in [3H]GBR 12783 binding which were significantly greater than decreases in [3H]cocaine binding. However, 50 mumol/l pHMBS produced a similar decrease in the Bmax of the specific binding of both radioligands. The pHMBS-induced reduction of [3H]GBR 12783 binding was not reversed by drugs whose action is purely that of uptake inhibition or by substrates of the dopamine carrier. Some of these drugs (100 mumol/l dopamine, 1 mumol/l mazindol or 100 mumol/l cocaine) protected the specific binding of [3H]cocaine against the effects of pHMBS, whereas 1 mmol/l p-tyramine, 10 mumol/l nomifensine and 10 nmol/l GBR 12783 were ineffective. Addition of 120 mmol/l Na+, 1 mmol/l Ca2+ or 10 mmol/l Mg2+ to a treatment medium containing 10 mmol/l Na+ significantly reduced the effects of pHMBS on the specific binding of both radioligands. When striatal cell membranes were treated in a medium containing 130 mmol/l Na+, there was a general decrease in the effects of ions on the reductions of specific binding produced by DTNB or pHMBS.(ABSTRACT TRUNCATED AT 400 WORDS)
我们比较了用离子型亲水性巯基试剂处理大鼠纹状体细胞膜对[³H]可卡因和[³H]GBR 12783（1-[2-(二苯甲氧基)乙基]-4-(3-苯基-2-[1-³H]丙烯基)-哌嗪）与多巴胺神经元转运体特异性结合的影响。用1 mmol/L 5,5'-二硫代双(2-硝基苯甲酸)(DTNB)处理导致两种放射性配体的特异性结合出现类似的时间和浓度依赖性降低。所测试的任何摄取阻滞剂都不能防止1 mmol/L DTNB的作用。向含有10 mmol/L Na⁺的处理介质中添加(亚)毫摩尔浓度的CaCl₂或MgCl₂，或250 mmol/L KCl，显著增加了DTNB诱导的两种放射性配体特异性结合的降低。阳离子可能是造成这种效应的原因，因为当添加1 mmol/L CaCl₂或50 - 250 mmol/L NaCl时，离子与DTNB结合会诱导类似的结合降低。阳离子对DTNB诱导的结合抑制作用通常对[³H]GBR 12783的影响比对[³H]可卡因结合的影响更明显。当添加到含有10 mmol/L Na⁺的介质中时，1 mmol/L DTNB导致两种放射性配体特异性结合的Bmax降低。添加1 mmol/L Ca²⁺维持或增加了这种Bmax降低，并引起亲和力下降，这对[³H]GBR 12783结合具有显著意义。用对羟基汞苯磺酸钠(pHMBS)的钠盐处理膜会导致[³H]GBR 12783结合出现时间和浓度依赖性降低，其降低幅度明显大于[³H]可卡因结合的降低幅度。然而，50 μmol/L pHMBS使两种放射性配体特异性结合的Bmax出现类似程度的降低。pHMBS诱导的[³H]GBR 12783结合降低不能被作用纯粹为摄取抑制的药物或多巴胺载体的底物所逆转。其中一些药物(100 μmol/L多巴胺、1 μmol/L吗茚酮或100 μmol/L可卡因)可保护[³H]可卡因的特异性结合免受pHMBS的影响，而1 mmol/L对酪胺、10 μmol/L诺米芬辛和10 nmol/L GBR 12783则无效。向含有10 mmol/L Na⁺的处理介质中添加120 mmol/L Na⁺、1 mmol/L Ca²⁺或10 mmol/L Mg²⁺，显著降低了pHMBS对两种放射性配体特异性结合的影响。当在含有130 mmol/L Na⁺的介质中处理纹状体细胞膜时，离子对DTNB或pHMBS产生的特异性结合降低的影响总体上有所减弱。(摘要截选至400字)
