家兎腎皮質切片에서의 有機酸移動 過程의 動力學的分析

Abstract

[한글]

[영문]

Many substances are added to talc urine by tubular secretion, that is, by transport from the peritubular fluid into the tubular lumen. Clearance studies in various species have served to identify several independent transport mechanisms, one being responsible for the secretion of a variety of weak organic acids, another for several organic bases (Taggart, 1958). The mechanism studied most extensively is that which secretes the various weak organic acids, such as phenosulfonphthalein dyes p-aminohippurate, Diodrast, Penicillin and Benemid.

Studies in several species indicate that the transport process is limited to the maximal convoluted tubule (Forster and Copenhaver, 1956; Malvin et al., 1958: Foulkes and Miller, 1959) and is dependent on the metabolic energy (Cross and Taggart, 1950; Mudge and Taggart, 1950; Taggart and Forster, 1950; Shideman et al., 1951; Forster find Copenhaver, 1956; Farah anti Rennick 1956). Maximal rates of transport(Tm) have been demonstrated for p-aminohippurate (Smith et al., 1942; Cho et al., 1960; Forster and Hong, 1962), Diodrast (Cho et al., 1960; Forster and Hong, 1962), and phenolsulfonphthalein(Shannon, 1935). Furthermore, the ability of the various compounds to compete with one another for transport has been demonstrated and is interpreted as the convincing evidence that they share a common secretory mechanism(Taggart, 1958; Lotspeich, 1959; Forster, 1951). Emphasis more recently has been placed on the differences in maximal transfer rates and it has been shown that the more slowly excreted compounds are relatively the more effective competitive inhibitors (Forster et al., 1957; Che 7t at., 1960; Fssig and Taggart, 1960; Forster and Hong, 1962). Furthermore, when two or more competing substances are Presented at the same time, their combined transfer rate is less than the sam of their individual transfer rates measured when they are presented singly(Forster and Hong, 1962), indicating that the graded affinities for the carrier(s) are related to dissociation constant of the individual competitors.

In view of these considerations, the present investigation was undertaken to systematically compare the inhibitory strength of various competitors on the uptake of p-aminohippurate(PAH) and phenolsulfonphthalein (PSP) by the rabbit renal cortical slice.

Male rabbits weighing, on the average, 2kg were used. Thin slices of rental cortex, approximatly 0.5 mm thick, were Prepared in chilled 0.13 M NaCl-0.02 M KCI Solution by means of a Stadie-Riggs microtome. Appoximatly 300 mg of slices were added to a large test tube (2×25cm) containing 5ml of the incubation medium and were incubated for 1 hour at 25℃ water bath under the constant oxygen supply. The electrolyte composition of the basic medium was identical with Cross-Taggart acetate medium (Cross and Taggart, 1960). For the kinetic analysis, the initial concentration of PSP or PAH in the medium was varied from 67 to 536μM/L while an inhibitor was added to the basic medium to a desired concentration. At the end of incubation period both the slices and media were analysed for PSP or PAH by the method of Forster and Copenhaver (1956) and by the method of Bratton anti Marshall modified by Smith et al. (1945), respectively.

In the first series of experiments, the rates of PSP and PAH uptake were studied while varying the concentration of PSP or PAH in the medium. In the next series, the effect of various organic acids such as Diodrast, Benemid, Decholate, Bromsulfalein(BSP), Bromcresol green(BCG), and Chlorphenol red (CPR) on the uptake

of PSP or PAH was studied, and the value of Ki for each inhibitor was computed by means of Michaelis-Menten analysis. For som inhibitors, their effects on the tissue respiration were also studied by using a Warburg manometer. Finally, the inhibition

of PAH uptake by the slices was studied presence of vatious organic acids at the concentration proportional to relative Ki ratio.

The results may be briefly summarized as fellows:

1. The overall accumulation of PSP or PAH displayed a typical active transport characteristics, the transport process being compatible with the Michaelis-Menten kinetics.

2. The values of velocity maxima(Vmax) for PAH and PSP transport were approximately 4.5 and 2.0 mM per kg wet tissue per hour, respectively, while the Michaelis constants (Km) were 0.58 and 0.17mM per liter, respectively.

3. Diodrast, Benemid and Decholate, all of which are well-known inhibitors of organic acid secretion in the kidney, competitively inhibited the accumulation of both PSP and PAH in the slices without affecting tissue respiration. In general, all of theme substances inhibited the accumulation of PAH more than that of PSP.

4. Organic acid dyes such as PSP, CPR, BCG, and BSP also inhibited tissue accumulation of PAH in a competitive manner.

5. The values of Ki which indicates the real strength of inhibition were 2.024, 0.364, 0.246, 0.226, 0.201, 0.102, 0.052 and 0.016 for PAH, Decholate, CPR, PSP, Diodrast, BSP, Benemid and BCG, respectively.

6. When the various organic acids were added to the incubation medium at the concentration pro-portional to the relative Ki value, the accumulation of PAH wart inhibited to the same extent.

These results indicate that the tubular secretions of various organic acids studied in this investigation are mediated by a certain transport carrier and the difference in the strength of competitive inhibition observed among various substances is due to the difference in the affinity of these substances to the

carrier(s).