Substrate utilization in the isolated perfused cortical thick ascending limb of rabbit nephron

Abstract

Isolated segments of cortical thick ascending limbs (cTAL) of rabbit kidney were perfused in vitro and the equivalent short circuit current (Isc) was measured. In a first series all substrates were removed on either side. Isc fell rapidly to 50 +/- 12% after 3 min and to 27 +/- 6% (n = 5) after 10 min. This indicates that in cTAL segments Isc is strictly dependent on the presence of substrates. In series two it was tested what substrates can be utilized by the cTAL segment, and from which epithelial side [bath (b) or lumen (1)] the substrates are taken up. From the 1-side only butyrate (10 mmol X 1(-1) sustained the Isc at 95 +/- 2% (n = 7). All other tested substrates (10 mmol X 1(-1): pyruvate, acetate, beta-OH-butyrate, D-glucose, and L-lactate lead to a marked decline in Isc. From the b-side several substrates (5--10 mmol X 1(-1) sustained the Isc: D-glucose, D-mannose, butyrate, beta-OH-butyrate, acetoacetate, L-lactate, acetate and pyruvate. Other compounds (1--10 mmol X 1(-1): citrate, alpha-ketoglutarate, succinate, glutamate, glutamine, propionate, caprylate and oleate did not sustain Isc. In the third series the mechanism of substrate utilization from the basolateral cell side was studied. It was shown that the Isc is a saturable function of the D-glucose, L-lactate, acetate, pyruvate or beta-OH-butyrate concentration with apparent Km's between 0.05--1.0 mmol X 1(-1). Several known inhibitors of sugar and of anion transport were tested at the bath side: phlorrhizin was without effect. Phloretin (500 mumol X 1(-1) inhibited Isc by 96%, yet its effect was not dependent on the presence of substrates on the b-side since inhibition occurred also if the b-perfusate contained no substrate and Isc was driven by luminal butyrate. Also SITS (5 mmol X 1(-1) exerted only a small inhibitory effect which was not specific since it was also observed with luminal butyrate. alpha-Cyano-m-OH-cinnamate (10 mmol X 1(-1) inhibited the Isc specifically when L-lactate was the bath substrate. Probenecid (1 mmol X 1(-1) had a similar yet less marked inhibitory effect. The D-glucose uptake from the b-side was specifically inhibited by cytochalasin B at 5 X 10(-6) mol X 1(-1). We conclude that the cTAL segment of the rabbit utilizes D-glucose and/or small anions such as pyruvate or L-lactate or acetate to energize salt reabsorption.(ABSTRACT TRUNCATED AT 400 WORDS)