We assessed the effects of antidiuretic hormone and cyclic adenosine monophosphate (cAMP) analogues on transepithelial voltage, Ve, and/or net chloride absorption in isolated mouse medullary (mTALH) and cortical (cTALH) thick ascending limbs of Henle; the passive NaCl permeability characteristics and electrical properties of the mTALH; and the effects of anion and cation substitutions and transport inhibitors on both basal and ADH-stimulated Ve and/or net chloride absorption in the mTALH. The data demonstrate that these two segments are functionally heterogeneous: ADH, at concentrations comparable to plasma levels seen in mammalian species during ordinary antidiuresis, and/or cAMP increase three- to fourfold the rate of NaCl absorption in the mTALH but not in the cTALH. The ion substitution and inhibitor data are consistent with the view that NaCl absorption in the mTALH depends on a secondary active transport process: NaCl entry across luminal membranes is a coupled process of indeterminate stoichiometry that is driven by the transmembrane electrochemical gradient for Na+, which is maintained by Na+-K+-ATPase. Finally, the data demonstrate that the mTALH is electrically leaky whether measured electrically, 11 omega . cm2, or isotopically, 50 omega . cm2, but essentially water impermeable; and that the mTALH is perm-selective for Na+ with respect to Cl-. The disparity between electrical resistances measured directly with respect to those calculated from tracer fluxes, together with the hybrid characteristics of mTALH junctional complexes (leaky to Na+ and Cl-; tight to water), may be reconciled by assuming that mTALH junctional complexes contain passive ion permeation pathways composed of narrow channels through which ions pass in single-file fashion.

We measured the relations between tubular perfusion rate and the rate of net NaCl transport in medullary thick ascending limbs of Henle (mTALH) either in the presence or absence of ADH. These data, together with the known Na+, Cl-, and water permeability characteristics of the mTALH, were used to calculate tau NaCl (mol . s-1 . cm-2), the rate of conservative Cl- transport from lumen through cells to interspaces; and CNaCl, the effective NaCl concentration in lateral intercellular spaces. The experimental results indicate that in these tubules the rate of net Cl- absorption increases monotonically with perfusion rate, and that at a given perfusion rate ADH increases the rate of net salt absorption. The theoretical calculations show clearly that the ADH-mediated increase in salt absorption depends on an increase in the rate of conservative transcellular Cl- transport. However, the present analytical data do not permit a distinction between wholly electroneutral apical membrane NaCl entry with respect to a process in which apical membrane Na+/Cl- entry has a stoichiometry less than unity, and electrogenic Na+ transport accounts for the remaining component of net Na+ absorption. Identification of the stoichiometry of the Na+/Cl- apical membrane entry step will depend, among other factors, on identifying explicitly the diffusion resistance of paracellular fluid and the mode of passive ion transport across junctional complexes.

A B S T R A C T This paper describes the inhibitory effect of prostaglandin E2 (PGE2) on antidiuretic hormone (ADH)-stimulated net Cl-absorption and spontaneous transepithelial voltage (Ve) in single medullary thick ascending limbs of Henle (TALH, thick ascending limb; mTALH, medullary segment; cTALH, cortical segment) obtained from mouse kidney. The experimental data indicate that PGE2 reduced the ADHdependent values of net Cl-absorption (Je,', eq cm-2 s-') and Ve (mV) in a dose-dependent manner; that increasing concentrations of peritubular ADH reversed the PGE2-mediated reductions in the ADH-dependent moiety of Ve in the mouse mTALH; that PGE2 had no effect on cyclic AMP-stimulated increments in Ve in the mouse mTALH; and that PGE2 had no In the renal medulla, this process enriches medullary osmolality, thereby contributing to urinary concentrating power (13,14). In certain species, like the mouse, salt absorption (11) and adenyl cyclase activity (15,16) in the water permeability of mammalian collecting ducts.In the isolated mouse mTALH, NaCl absorption involves the coupled entry of Cl-, and possibly K+ (17, 18), to Na+ entry across apical plasma membranes. In other words, Cl-transport in this nephron segment is a secondary active transport process, driven in part by the Na+ gradient between luminal fluid and cell interior (11,17). Conservative NaCl absorption in this nephron segment is enhanced considerably, and in a dose-dependent manner, by ADH, operating through the second messenger cyclic (c)AMP (11). Conversely, conservative NaCl absorption in the mouse mTALH is suppressed significantly by increases in peritubular osmolality independently of transepithelial osmotic pressure gradients (19).The mouse mTALH, like the rabbit cTALH (6, 20) and mTALH (7), behaves as a hybrid epithelium: the mouse mTALH is electrically leaky (11, 17) and rather permeable to Na+ and to 17), yet is virtually water impermeable either in the presence or absence of ADH (9-11). Consequently, as medullary peritubular salt concentrations vary, there occur varying degrees of dissipative NaCl backleak from interstitium to lumen through the paracellular pathway. Thus, for the mouse mTALH, maintenance of diluting power in the presence of varying NaCl concentrations in peritubular media depends, at least in part, on the interplay between ADH and peritubular osmolality in modifying conservative NaCl transport (11, 19). We will argue in this paper that PGE2 represents yet another factor that modulates the latter in the mouse mTALH.In the amphibian urinary bladder, Orloff et al. (21) found that prostaglandins inhibited the hydro6smotic effect of ADH but did not change the response of the epithelium to cAMP. They inferred that prostaglandins had acted at a locus proximal to hormone-dependent accumulation of cAMP within the cell, with little or no discernible prostaglandin action on transport events beyond cAMP accumulation. Based on in vitro studies in the isolated rabbit collecting duct, Grantham and Orloff (22) also suggested that prosta...

We evaluated the effects of increasing bath osmolality on both the passive permeability properties and the ADH-dependent rates of net Cl- absorption in isolated mouse medullary thick ascending limbs of Henle (mTALH). Increases in both osmolality to 900 mosmol/kg H2O with 600 mM urea had no effect on either the electrical (PNa/PCl ratio, 1.7 and 1.9 with and without peritubular urea, respectively) or tracer (PNa, 0.21 and 0.22 micrometers . s-1 with and without peritubular urea, respectively) ionic permeability characteristics of the mTALH. However, this degree of urea bath hypertonicity reduced reversibly both JnetNaCl, the net rate of transepithelial NaCl absorption, and Ve, the spontaneous transepithelial voltage: JnetNaCl fell by 85% and Ve by 70%. Both of these latter effects could be accounted for quantitatively by an 85% reduction in tau NaCl, the rate of conservative transcellular NaCl transport. The inhibition of Ve by peritubular medium urea hypertonicity was not altered by supramaximal bath concentrations of ADH, supramaximal bath concentrations of cAMP analogues, or symmetrical addition of urea to perfusate and bath. Increases in peritubular medium mannitol concentrations also reduced Ve; the inhibition of Ve was not reversed by supramaximal bath concentrations of aDH. Cell volume remained unchanged with peritubular urea but was reduced by peritubular mannitol. These data indicate that in the mTALH increases in bath osmolality with nonelectrolytes inhibit tau NaCl noncompetitively with respect to ADH or cAMP and independently of cell volume. JnetCl was also reduced with increases in peritubular medium NaCl concentration and was associated with a reduction in cell volume.

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