INTRACELLULAR-DISTRIBUTION OF CYSTINE IN CYSTINE-LOADED PROXIMAL TUBULES

Cellular cystine loading with cystine dimethyl ester has been shown to inhibit transport in proximal convoluted tubules perfused in vitro an d decrease the rate of oxygen consumption in suspensions of proximal t ubules. The present study was designed to examine the intracellular di stribution of cystine in this model of the Fanconi syndrome of cystino sis and to determine whether cystine or its degradation product, cyste ine, is the cytotoxic agent in cystine-loaded rabbit proximal tubules. Tubules were incubated with 2 mmol/L cystine dimethyl ester for 10 mi n at 37 degrees C and subjected to cellular fractionation. The intraly sosomal cystine content (272 +/- 125 nmol/mg protein) was significantl y higher than that measured in the nucleus (8.7 +/- 2.0 nmol/mg protei n) and cytosol (9.8 +/- 4.0 nmol/mg protein (p < 0.05). Electron micro graphs of tubules loaded with cystine depicted large swollen lysosomes . To determine whether cystine or its breakdown product, cysteine, was the cytotoxic agent in tubules incubated with cystine dimethyl ester, the intracellular cystine and cysteine contents were measured and fou nd to be 86.5 +/- 14.8 and 5.7 +/- 1.7 nmol/mg protein, respectively. These tubules had a 50% decrease in the rate of O-2 consumption. To ex amine whether the increased level of intracellular cysteine played a r ole in this decrease in O-2 consumption, we loaded tubules with 2 mmol /L cysteine methyl ester for 10 min. Despite an intracellular cysteine concentration of 59.6 +/- 5.8 nmol/mg protein, cysteine-loaded tubule s had a rate of O-2 consumption equal to that measured in control tubu les. Thus, intracellular cystine loading significantly increases intra lysosomal cystine content. The inhibition of tubular respiration with cystine dimethyl ester is due to cystine rather than its degradation p roduct, cysteine.