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JA Near, JC Bigelow and RM Wightman
Department of Chemistry, Indiana University, Bloomington.
Uptake of dopamine (DA) by chopped tissue prepared from rat corpus striatum has been examined to determine whether one or two kinetically distinct uptake sites exist. Two methods were used: direct measurement of accumulated [3H]DA and determination of the rate of formation of 3,4- dihydroxyphenylacetic acid (DOPAC) after exposure to DA. The rate of formation of DOPAC in the latter experiments is a direct function of the rate of DA uptake. The rates of [3H]DA uptake and DOPAC formation are both linear with time in the presence of 10 microM substrate. Studies of [3H]DA accumulation into chopped tissue reveal two apparent components with Km values of 160 nM and 3.8 microM, whereas similar experiments with striatal homogenate or synaptosomes yield a single uptake component with a Km equivalent to the lower value found in chopped tissue. Evaluation of DA uptake via the rate of DOPAC formation gave a Km value of 2.3 microM. (High substrate values were used, so a lower value for Km is not apparent in the data.) The high Km-value component was absent in animals with a lesioned striatum induced by prior nigral injections of 6-hydroxydopamine. Several pharmacologic agents (benztropine, amfonelic acid, bupropion, nomifensine and ouabain) were evaluated. All reduced the uptake of DA in chopped tissue, but with reduced potencies compared with the effect of these agents in synaptosomes. The high Km activity in chopped tissue, as well as the apparent reduced potency of uptake inhibitor, appears to arise from the diffusional barrier present inside more intact tissue. This barrier is not present in homogenates or synaptosomes, and, thus, a single uptake process is seen.
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