Coupling of metabolic CO₂ production to ion transport in isolated rat thick ascending limbs and collecting tubules

Abstract

Metabolic CO₂ production from appropriate [U-¹⁴C]-labelled substrates (eitherl-lactate ord-glucose) was measured in single pieces of tubule as previously described (Le Bouffant et al. 1984). Changing the incubate osmotic pressure by mannitol addition resulted in an increase in oxidative metabolism which was more marked in outermedullary segments (MAL and MCT) than in cortical segments (CAL and CCT). Availability of metabolic substrate was not rate limiting under these conditions because FCCP addition (1 μmol·l⁻¹) produced a marked rise in CO₂ production in these structures.

Ouabain (1 mmol·l⁻¹) decreased by more than 50% the CO₂ production by CAL, MAL, CCT and MCT samples, indicating that the larger part of oxidative metabolism was coupled to active Na transport. Furosemide addition (10⁻⁵ mol·l⁻¹) to CAL and MAL samples, or amiloride addition (10⁻⁴ mol·l⁻¹) to CCT and MCT samples reduced the rate of CO₂ production to an extent almost similar to that obtained with ouabain, an observation suggesting that apical entry of Na⁺ was present in these non-perfused tubules.

Finally, the effects of changing the concentration of either K⁺ or Cl⁻ was tested in CAL samples. K⁺ suppression greatly depressed the rate of CO₂ production. Replacement of chloride with sulfate also decreased this rate to an extent similar to that observed with furosemide. The CO₂ production increased in a sigmoid way (apparentK _a=41 mmol·l⁻¹, Hill coefficient=2.12) as a function of [Cl⁻] in the incubate, suggesting that oxidative metabolism was coupled to bath chloride via the Cl⁻-requiring Na entry along the 1 Na⁺−1K⁺−2Cl⁻ luminal contrasport system.