Holz R W
Proc Natl Acad Sci U S A. 1978 Oct;75(10):5190-4. doi: 10.1073/pnas.75.10.5190.
The effects of ATP, Mg(2+), and various agents on pH gradient, membrane potential, and catecholamine transport across membranes of intact bovine chromaffin vesicles were investigated. Methylamine and thiocyanate (SCN(-)) distributions across the vesicle membrane were used to estimate the H(+) concentration gradient and membrane potential, respectively. The H(+) concentration ratio (intravesiculanmedium) equals 16 when the medium pH is 6.9 and is unaltered by ATP and Mg(2+). In the absence of ATP and Mg(2+), the steady-state intravesicular S(14)CN(-) concentration is lower than the medium concentration. ATP and Mg(2+) cause an increased influx and a decreased efflux of SCN(-) that results in SCN(-) being concentrated in the vesicles 6- to 8-fold over the medium. The findings are consistent with an ATP,Mg(2+)-induced potential of approximately 50 mV (intravesicular side positive). Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), a H(+) translocater, and N-ethylmaleimide (NEM), a sulfhydryl reagent, decrease the SCN(-) ratio and, thus, the membrane potential in the presence of ATP and Mg(2+). They have no effect on the H(+) concentration gradient. The rate of catecholamine uptake into vesicles is increased 4- to 6-fold by ATP and Mg(2+). The ATP,Mg(2+)-stimulated uptake is inhibited by FCCP and NEM over the same concentration ranges that reduce the SCN(-) distribution (membrane potential). FCCP increases and NEM decreases vesicular membrane ATPase activity. Thus, catecholamine uptake is correlated to an inside-positive membrane potential, and not to ATPase activity. If catecholamine uptake is coupled to membrane potential, then a charged species must be involved in the transport mechanism. Reserpine and rotenone inhibit catecholamine influx but have no effect on the H(+) electrochemical gradient; they probably act at a step before coupling to the membrane potential (or the H(+) electrochemical gradient). Atractyloside, an inhibitor of nucleotide transport, has no effects on catecholamine transport or the H(+) electrochemical gradient.
研究了ATP、Mg(2+)和各种试剂对完整牛嗜铬小泡跨膜的pH梯度、膜电位和儿茶酚胺转运的影响。分别用甲胺和硫氰酸盐（SCN(-)）在小泡膜两侧的分布来估计H(+)浓度梯度和膜电位。当培养基pH为6.9时，H(+)浓度比（小泡内/培养基）等于16，且不受ATP和Mg(2+)的影响。在没有ATP和Mg(2+)的情况下，稳态时小泡内S(14)CN(-)浓度低于培养基浓度。ATP和Mg(2+)导致SCN(-)的流入增加和流出减少，结果是SCN(-)在小泡中的浓度比培养基高6至8倍。这些发现与ATP、Mg(2+)诱导的约50 mV的电位一致（小泡内侧为正）。羰基氰化物对三氟甲氧基苯腙（FCCP），一种H(+)转运体，和N-乙基马来酰亚胺（NEM），一种巯基试剂，在有ATP和Mg(2+)存在时降低SCN(-)比值，从而降低膜电位。它们对H(+)浓度梯度没有影响。ATP和Mg(2+)使儿茶酚胺摄入小泡的速率增加4至6倍。在降低SCN(-)分布（膜电位）的相同浓度范围内，FCCP和NEM抑制ATP、Mg(2+)刺激的摄取。FCCP增加而NEM降低小泡膜ATP酶活性。因此，儿茶酚胺摄取与内侧为正的膜电位相关，而与ATP酶活性无关。如果儿茶酚胺摄取与膜电位偶联，那么一种带电物质必定参与转运机制。利血平和鱼藤酮抑制儿茶酚胺流入，但对H(+)电化学梯度没有影响；它们可能作用于与膜电位（或H(+)电化学梯度）偶联之前的步骤。核苷酸转运抑制剂苍术苷对儿茶酚胺转运或H(+)电化学梯度没有影响。
