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1. 1. Viable cells were isolated from the thick ascending limb of Henle's loop (TALH) of rabbit renal medulla. 2. 2. The TALH cells were characterized by high activities of calcitonin-stimulated adenylate cyclase and Na-K-ATPase and low activities of alkaline phosphatase, γ-glutamyl-transpeptidase and ADH-sensitive adenylate cyclase. 3. 3. The oxygen consumption of the TALH cells was inhibitable in a dose dependent manner by furosemide and bumetanide. 4. 4. Ouabain (10 −4 M) also inhibited oxygen consumption as did replacement of either Na or Cl in the incubation medium. 5. 5. Preliminary experiments with plasma membrane vesicles prepared from the TALH cells indicated the presence of a coupled NaCl transport system.

A homogeneous population of single cells from the thick ascending limb of Henle's loop (TALH) has been isolated from the rabbit kidney medulla. A total medullary cell suspension was prepared by a series of collagenase, hyaluronidase, and trypsin digestions and separated on a Ficoll gradient (2.6-30.7% wt/wt). Morphologically, the cells isolated from the TALH were homogeneous and showed polarity within their plasma membrane structure, with a few blunt microvilli on their apical surface and deep infoldings of the basal-lateral membrane. Biochemically, the TALH cells were highly enriched in calcitonin-sensitive adenylate cyclase and Na, K-ATPase. Alkaline phosphatase and arginine vasopressin-sensitive adenylate cyclase, highly concentrated in proximal tubule and collecting duct, were present only in low concentrations in the TALH cells. Additionally, furosemide, a diuretic inhibiting sodium chloride transport in the TALH in vivo, inhibited oxygen consumption of the TALH cells in a dose-dependent manner. The TALH cells were viable, as judged by morphological appearance, trypan blue exclusion, the response of oxygen consumption to 2,4-dinitrophenol, succinate and ouabain, and the cellular Na, K and ATP levels.

A membrane fraction, rich in basal-lateral plasma membranes, was prepared from the rectal gland of the spiny dogfish,Squalus acanthias, and the uptake of22Na into the plasma membrane vesicles was investigated by a rapid filtration technique. Sodium uptake was greatest in the presence of a chloride gradient directed into the vesicles; it was strikingly reduced when chloride was replaced with nitrate and was even slower with gluconate. If the membrane vesicles were pre-equilibrated with potassium chloride or potassium nitrate plus valinomycin, to minimize any electrical driving forces on sodium movement, the uptake of sodium was still greatest in the presence of chloride and remarkably decreased in the presence of nitrate. Furosemide, 10−3 and 10−4 M, decreased sodium uptake into the vesicles in a dose dependent manner only in the presence of chloride. Furthermore, saturation of sodium uptake by increasing sodium chloride concentrations was observed. The above results provide direct evidence for a coupling of sodium and chloride fluxes across the plasma membrane of the rectal gland via a cotransport system sensitive to furosemide. They support the hypothesis that chloride secretion of the rectal gland is a secondary active transport and is driven by the sodium gradient across the basal-lateral membranes of the cell.

This review discusses the activation of transport pathways during volume regulation, including their characteristics, the possible biochemical pathways that may mediate the activation of transport pathways, and the relations between volume regulation and transepithelial transport in renal cells. Many cells regulate their volume when exposed to an anisotonic medium. The changes in cell volume are caused by activation of ion transport pathways, plus the accompanying osmotically driven water movement such that cell volume returns toward normal levels. The swelling of hypertonically shrunken cells is termed regulatory volume increase (RVI) and involves an influx of NaCl into the cell via either activation of Na-Cl, Na-K-2Cl cotransport systems, or Na+-H+ and Cl(-)-HCO3- exchangers. The reshrinking of hypotonically swollen cells is termed regulatory volume decrease (RVD) and involves an efflux of KCl and water from the cell by activation of either separate K+ and Cl-conductances, a K-Cl cotransport system, or parallel K+-H+ and Cl(-)-HCO3- exchangers. The biochemical mechanisms involved in the activation of transport systems are largely unknown, however, the phosphoinositide pathway may be implicated in RVI; phorbol esters, cGMP, and Ca2+ affect the process of volume regulation. Renal tubular cells, as well as the blood cells that traverse the medulla, are subjected to increasing osmotic gradients from the corticomedullary junction to the papillary tip, as well as changing interstitial and tubule fluid osmolarity, depending on the diuretic state of the animal. Medullary cells from the loop of Henle and the papilla can volume regulate by activating Na-K-2Cl cotransport or Na+-H+ and Cl(-)-HCO3- exchange systems. Both Na-Cl and Na-K-2Cl cotransport systems have been identified in the medullary Loop of Henle and it is postulated that the Na-K-2Cl cotransport system predominates during RVI and affects transepithelial NaCl transport while the Na-Cl cotransport system may function during RVD in these cells.

Brush-border membrane vesicles were isolated from the intestine and kidney of the winter flounder,Pseudopleuronectes americanus, and the transport ofd-glucose,l-alanine and sodium was examined by a rapid filtration technique.d-glucose,l-alanine, and sodium entered the same osmotically reactive space suggesting that uptake into vesicles represents transport across rather than binding to the membrane. d-glucose andl-alanine uptake by intestinal and renal brush-border membrane vesicles was stimulated by sodium as compared to potassium or choline. In the presence of a sodium chloride gradient, overshooting uptake was observed indicating a transient intravesicular accumulation ofd-glucose andl-alanine. The sodium-dependentd-glucose uptake was inhibited by phlorizin andd-galactose while the transport ofl-alanine was inhibited byl-phenylalanine. The sodium-dependent transport ofd-glucose andl-alanine was affected by the electrical potential difference across the vesicle membrane; the addition of valinomycin in the presence of an inwardly directed potassium chloride gradient inhibited sodium-dependent solute uptake, whereas replacing chloride or gluconate with more permeant anions, such as SCN−, stimulated uptake. Similar results were obtained with intestinal and renal membranes; they document the presence of sodium/d-glucose and sodium/l-alanine cotransport systems in the brush-border membrane of intestine and kidney. Sodium uptake into brush border membrane vesicles from the flounder intestine and kidney was saturable (tracer replacement) and trans-stimulated (tracer coupling), indicating transport via facilitated diffusion systems. Additionally, sodium uptake was only slightly affected by superimposing diffusion potentials demonstrating that the majority of sodium transport was by electroneutral coupled processes. In both the intestinal and kidney brush-border membrane vesicles sodium uptake was inhibited by an inwardly directed proton gradient suggesting the presence of a sodium/proton exchange mechanism. In intestinal, but not in renal membrane preparations, sodium uptake was stimulated by chloride. Chloride stimulation was abolished after preincubation with furosemide indicating the presence of an additional coupled sodium-chloride transport in the intestinal brush-border membranes.

The present studies examined the mechanism of bicarbonate transport across basolateral membrane vesicles prepared from rabbit renal cortex. Isotopic sodium uptake was stimulated by bicarbonate when compared with gluconate (2.5 nmol/mg protein per 5 s versus 1.4 nmol/mg protein per 5 s), and this process was inhibited by disulfonic stilbenes. Imposition of an interior-positive potassium diffusion potential further stimulated isotopic sodium uptake to 3.4 nmol/mg protein per 5 s, an effect that occurred only in the presence of bicarbonate and was blocked by disulfonic stilbenes. Kinetic analysis of the rate of bicarbonate-dependent sodium uptake as a function of sodium concentration revealed saturable stimulation with a Vmax of 2.7 nmol/mg protein per 2 s and a Km of 10.4 mM. The effect of bicarbonate concentration on bicarbonate-dependent sodium uptake was more complex. The present results demonstrate an electrogenic (negatively charged) sodium/bicarbonate cotransporter in basolateral membrane vesicles from the rabbit renal cortex. The electrogenicity implies a stoichiometry of at least two bicarbonate ions for each sodium ion.

The binding of phenol red to the microsomal fraction of rabbit kidney cortex was rapid, reversible and consisted of two independent populations of binding sites: a high affinity and low capacity class which had an association constant of 11.29 · 103 M−1 and a binding capacity of 2.41 μmol phenol red bound per g of protein, and a low affinity binding population with an association constant of 0.80 · 103 M−1 and a maximal binding capacity of 55.06 μmol per g of protein. Probenecid (0.32 mM) competitively inhibited phenol red binding to only the high affinity binding site, whereas 2,4-dinitrophenol (0.77 mM) competitively inhibited phenol red binding to both the high and the low affinity population of binding sites. The binding of phenol red was highly sensitive of the cationic composition of the medium. The affinity of phenol red to the high and the low affinity binding populations was lowered by decreasing the sodium and potassium concentrations to 19 and 6 mequiv./1, respectively; however, the maximal binding capacity was unchanged. Calcium appeared to have no effect on the phenol red binding to the microsomes. All of these considerations suggest that the high affinity phenol red binding to the microsomal fraction may represent the interaction of phenol red with the physiological receptor necessary for organic acid transport at the peritubular membrane. Phenol red binding to the low affinity binding population may indicate an intracellular binding population which contributes to the intracellular accumulation of weak organic acids.

Isolated cells were prepared from the medullary thick ascending limb of Henle's loop (TALH) and the response of oxygen consumption was correlated with the active chloride transport system found in these cells. Oxygen consumption was 31.6 microliters O2/mg protein . h and inhibited 50% by the absence of either sodium or chloride in the incubation medium. The absence of both sodium and chloride produced no further inhibition of oxygen consumption. Ouabain (10(-4) M) inhibited oxygen consumption by 50% and the inhibitory effect depended on the presence of both sodium and chloride in the incubation medium. Further, furosemide inhibited oxygen consumption by a maximum of 50% at 10(-3) M and also had no inhibitory effect if either sodium or chloride were absent. Furosemide had no effect on the Na, K-ATPase activity or ATP levels of the TALH cells. Thus, the data suggest or ATP levels of the TALH cells. Thus, the data suggest that 50% of the oxygen consumption of the TALH cells is related to the movement of sodium and chloride into the cell and that the ions may be transported in a coupled manner. In addition the effect of various diuretics on oxygen consumption in the isolated TALH cells was tested. The diuretics could be grouped in three categories: (1) highly effective in inhibiting chloride-dependent oxygen consumption with an apparent inhibitory constant (Ki) of around 10(-6) M, including the diuretics furosemide, bumetanide, ethacrynic acid-cysteine and piretanide, (2) diuretics which were less effective in inhibiting oxygen consumption with an apparent Ki of around 10(-4) M, HOE 740 and ethacrynic acid, and (3) diuretics which were ineffective in inhibiting chloride-dependent oxygen consumption, amiloride and hydrochlorothiazide.

p-Aminohippuric acid (PAH) transport was investigated in brush border vesicles isolated from renal proximal tubules of the winter flounder. Three characteristics of carrier-mediated transport were demonstrated: 1) unlabeled PAH inhibited the uptake of [3H]PAH; 2)[3H]PAH efflux from the vesicles was stimulated in the presence of unlabeled PAH in the extravesicular medium; and 3) PAH influx was inhibited by 2,4-dinitrophenol (DNP) and 4-acetamido-4'-isothiocyano-2,2'-disulfonic stilbene (SITS). D-Glucose plus a sodium gradient stimulated PAH uptake, as did a K2SO4 gradient plus valinomycin, suggesting that PAH is transported as an anion. In contrast, PAH uptake into a membrane fraction containing mainly basal-lateral plasma membranes exhibited a larger inhibition by probenecid but a smaller inhibition by unlabeled PAH and SITS. Thus, carrier-mediated transfer of PAH driven by the electrochemical potential difference for PAH is demonstrated in the brush border membrane of the flounder kidney.

The KCl cotransporter in the basolateral membrane of renal tubules may play a central role in the transcellular transport of NaCl. Because this transporter is electrically neutral, and also functions in parallel to the electrogenic Na,K-ATPase, there is an imbalance in charge which must be expressed as a cationic current across the basolateral membrane. Therefore, other pathways must also function in the basolateral membrane which permit the conductive exit of K+ in addition to the electrically-neutral KCl cotransporter. Another functional role for the KCl cotransporter is manifest during the cell volume regulatory response to cell swelling. In this setting (regulatory volume decrease), it appears that both electrically-neutral and electrically-coupled KCl efflux pathways are acutely activated. Very little is known at present about the mechanisms of short and long term regulation of the KCl cotransporter. A major obstacle at this point is the lack of a suitable, potent (that is, microM range) specific inhibitor of this transporter. It also appears that the chloride transport systems in basolateral membrane vesicles may be greatly influenced by the precise details of the method of preparation. Once these experimental details are mastered, and a suitable high affinity inhibitor is identified, then the detailed characterization and identification of the KCl cotransporter can be undertaken.

Specific binding of radiolabeled inhibitor was employed to localize the Na-pump sites (Na,K-ATPase) in rectal gland epithelium, a NaCl-secreting osmoregulatory tissue which is particularly rich in pump sites. Slices of gland tissue from spiny dogfish were incubated in suitable [3H]ouabain-containing media and then prepared for Na,K-ATPase assay, measurement of radiolabel binding, or quantitative freeze-dry autoradiography at the light microscope level. Gross freezing or drying artifacts were excluded by comparison with additional aldehyde-fixed slices. Characterization experiments demonstrated high-affinity binding which correlated with Na,K-ATPase inhibition and half-saturated at approximately 5 microM [3H]ouabain. At this concentration, the normal half-loading time was approximately 1 h and low-affinity binding to nonspecific sites was negligible. Autoradiographs from both 1- and 4-h incubated slices showed approximately 85% of the bound [3H]ouabain to be localized within a 1-micrometer wide boundary region where the highly infolded basal-lateral cell membrane are closest to the mitochondria. These results establish that most of the enormous Na,K-ATPase activity associated with rectal gland epithelium is in the basal-lateral cell membrane facing interstitial fluid and not in the luminal membrane facing secreted fluid. Moreover, distribution along the basal-lateral membrane appears to be nonuniform with a higher density of enzyme sites close to mitochondria.

Because the medullary thick ascending limb of the loop of Henle is the target of several polypeptide hormones that stimulate adenyl cyclase in this nephron segment, we examined the effects of cyclic AMP on thick ascending limb of the loop of Henle cells isolated by enzymatic digestion and density gradient centrifugation from the outer medulla of the rabbit kidney. The functional parameter that was measured was transport dependent oxygen consumption. Oxygen consumption was measured using a polarographic oxygen electrode in a constant temperature chamber. We found that dibutyryl cyclic AMP inhibited oxygen consumption in a dose dependent way. Maximal inhibition was observed at a concentration of 10−5M. The effect of dibutyryl cyclic AMP was not present in the absence of either sodium, chloride or both implying that its effect is restricted to the sodium and chloride dependent oxygen consumption. The effect of dibutyryl cyclic AMP was additive to that of furosemide·10−4M while that of furosemide was not additive to that of cyclic AMP suggesting that the site of action of cyclic AMP is distal to that of furosemide. The effect of dibutyryl cyclic AMP was not additive to that of ouabain and was absent in cells where oxygen consumption was stimulated with amphotericin B in the absence of chloride indicating that it has no effect on Na−K-ATPase. On the basis of these results, and by analogy with other epithelia where the effect of cyclic AMP on chloride transport has been demonstrated to be at the chloride conductance, the effect of cyclic AMP on transport dependent oxygen consumption can be most probably localized to the chloride conductance. The hormone responsible for the primary step was not identified in these experiments.

p -Aminohippuric acid (PAH) uptake was studied in basal-lateral membrane vesicles prepared from rabbit renal cortex. An outwardly directed hydroxyl gradient (pH o = 6.0, pH i = 7.6) stimulated PAH uptake slightly over that when the internal and external pH values were equal at 7.6. A 100 mM sodium gluconate gradient directed into the basal-lateral membrane vesicles increased PAH uptake about 2-fold over that when N- methyl- d -glucamine or potassium gluconate gradients were present. When hydroxyl and sodium gradients were simultaneously imposed (pH o = 6.0, pH i = 7.6 and 100 mM sodium gluconate extravesicularly) PAH uptake was stimulated greater than with the pH or Na + gradient alone. In fact, an ‘overshoot’ was observed. Countertransport experiments showed that either intravesicular PAH or intravesicular PAH and Na + could stimulate 3 H-PAH uptake. Probenecid, an inhibitor of organic anion transport, inhibited both the hydroxyl-stimulated and Na + gradient-stimulated PAH uptake but the greatest inhibition by probenecid was seen when the hydroxyl and sodium gradients were both present. Thus, it is proposed that the driving force for PAH accumulation across the basal-lateral membrane of the proximal tubule is a transport system which moves Na + and PAH into the cell for an hydroxyl ion leaving the cell, i.e. a sodium-dependent anion-anion exchange system.