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キーワード:
Brush border membrane; 2-Oxoglutarate; Succinate; Citrate; Lithium
要旨:
The transport of dicarboxylic acid in the late proximal convolution was investigated by measuring the 3.5 s effux of 2-oxoglutarate from the tubular lumen of rats starved for 3 days. The 3.5 s efflux of 2-oxoglutarate comprises two components, one due to movement across the brush border, obeying Michaelis Menten kinetics with an apparent Km of 0.13 mmol/l and a Jmax of 0.41 pmol cm−1 s−1 and the other due to diffusional movement presumably via the paracellular pathway with a permeability of 23.0 μm2 s−1.
Omission of sodium from the perfusion fluid reduced the transcellular efflux of 2-oxoglutarate by 76%, indicating a sodium-dependent transport system. Addition of 5 mmol/l lithium to the liminal and capillary perfusate reduced it by 56% indicating a specific inhibitory effect of lithium on dicarboxylic acid transport. Addition of 5 mmol/l H2DIDS to the luminal perfusate reduced 3.5s transcellular 2-oxoglutarate effux by 35%.
The molecular specificity of the system was assessed by studying the inhibitory effects of a series of dicarboxylates, both aliphatic and aromatic, on the 3.5 s efflux of 2-oxoglutarate. Inhibitory constants (apparent Ki) were calculated for comparative purposes assuming competitive inhibition. From this, the system was found to have optimal affinity for dicarboxylates in the trans-configuration with a four or five carbon chain (i.e. succinate and glutarate). Substitution on the 2-carbon atom with CH3−,OH−,SH−, and O=resulted in little reduction in inhibitory potency as compared to succinate litself. However, 2-substitution with NH+3 (not with N-acetyl) as well as 2,3-disubstitution with CH3−, OH− or SH−, strongly reduced or abolished the inhibitory potency. Only with the exception of pyruvate all monocarboxylates tested, did not inhibit 2-oxoglutarate transport, but all tricarboxylates tested, i.e. citrate, isocitrate and tricarballate had an inhibitory effect. Citrate inhibition was higher at acidic than alkaline pH.
A number of aromatic compounds was also tested. In most cases the inhibitory potency of the aromatic compounds was considerably weaker than that of the effective 4–5 carbon chain aliphatic compounds. Only benzene-1,4-dicarboxylate, benzene-2-nitro-1,4-dicarboxylate, and benzene-1,2-diacetate had a high inhibitory potency. In the case of the aromatic dicarboxylates the most important feature relating molecular structure to transport was the distance between the two carboxyl-groups in the molecule. It was further found that the presence of a nitrogen group in the ring shifted increasing inhibitory potency toward compounds with a smaller distance between the two carboxylic groups.
The data indicate a Na+-dependent transport system for dicarboxylic acids in the brush border of the proximal tubule with a wide specificity for Krebscycle intermediates and certain of their analogues. Other dicarboxylates are also transported by the system though with a significantly reduced potency. The system has also an affinity for lithium, pyruvate, paraaminohippurate, H2DIDS, and taurocholate.
