Pre-steady-state analysis of the turn-on and turn-off of water permeability in the kidney collecting tubule

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Abstract
Water transport across the mammalian collecting tubule is regulated by vasopressin-dependent water channel insertion into and retrieval from the cell apical membrane. The time course of osmotic water permeability (P f ) following addition and removal of vasopressin (VP) and 8-Br-cAMP was measured continuously by quantitative fluorescence microscopy using an impermeant fluorophore perfused in the lumen. Cortical collecting tubules were subjected to a 120 mOsm bath-to-lumen osmotic gradient at 37°C with 10–15 nl/min lumen perfusion and 10–20 ml/min bath exchange rate. With addition of VP (250 μU/ml), there was a 23±3 sec (sem,n=16) lag in whichP f did not change, followed by a rise inP f (initial rate 1.4±0.2×10−4 cm/sec2) to a maximum of 265±10×10−4 cm/sec. With addition of 8-Br-cAMP (0.01–1mm) there was an 11±2 sec lag. For [8-Br-cAMP]=0.01, 0.1 and 1mm, the initial rate ofP f increase following the lag was (units 10−4 cm/sec2): 1.1±0.1, 1.2±0.1 and 1.7±0.3. MaximumP f was (units 10−4 cm/sec): 64±4, 199±9 and 285±11. With removal of VP,P f decreased to baseline (12×10−4 cm/sec) with aT 1/2 of 18 min; removal of 0.1 and 1mm 8-Br-cAMP gaveT 1/2 of 4 and 8.5 min. These results demonstrate (i) a brief lag in theP f response, longer for stimulation by VP than by 8-Br-cAMP, representing the transient build-up of biochemical intermediates proximal to the water channel insertion step, (ii) similar initialdP f /dt (water channel insertion) over a wide range of [8-Br-cAMP] and steady-stateP f values, and (iii) more rapidP f decrease with removal of 8-Br-cAMP than with VP. These pre-steady-state results define the detailed kinetics of the turn-on and turn-off of tubuleP f and provide kinetic evidence that the rate-limiting step for turn-on ofP f is not the step at which VP regulates steady-stateP f . If water channel insertion is assumed to be the rate-limiting step in the turn-on ofP f , these results raise the possibility that water channels must be activated following insertion into the apical membrane.