Relationship between cell volume and ion transport in the early distal tubule of the Amphiuma kidney

The roles of apical and basolateral transport mechanisms in the regulation of cell volume and the hydraulic water permeabilities (Lp) of the individual cell membranes of the Amphiuma early distal tubule (diluting segment) were evaluated using video and optical techniques as well as conventional and Cl-sensitive microelectrodes. The Lp of the apical cell membrane calculated per square centimeter of tubule is less than 3% that of the basolateral cell membrane. Calculated per square centimeter of membrane, the Lp of the apical cell membrane is less than 40% that of the basolateral cell membrane. Thus, two factors are responsible for the asymmetry in the Lp of the early distal tubule: an intrinsic difference in the Lp per square centimeter of membrane area, and a difference in the surface areas of the apical and basolateral cell membranes. Early distal tubule cells do not regulate volume after a reduction in bath osmolality. This cell swelling occurs without a change in the intracellular Cl content or the basolateral cell membrane potential. In contrast, reducing the osmolality of the basolateral solution in the presence of luminal furosemide diminishes the magnitude of the increase in cell volume to a value below that predicted from the change in osmolality. This osmotic swelling is associated with a reduction in the intracellular Cl content. Hence, early distal tubule cells can lose solute in response to osmotic swelling, but only after the apical Na/K/Cl transporter is blocked. Inhibition of basolateral Na/K ATPase with ouabain results in severe cell swelling. This swelling in response to ouabain can be inhibited by the prior application of furosemide, which suggests that the swelling is due to the continued entry of solutes, primarily through the apical cotransport pathway.     

Urate transport across the apical membrane of renal proximal tubules

Since the molecular cloning of the renal apical urate/anion exchanger URAT1 (SLC22A12), several membrane proteins relevant to urate transport have been identified. In addition, the identification of PDZ (PSD-95, DglA, and ZO-1) domain protein PDZK1 as a binding partner of URAT1, and the emerging role of PDZ scaffold for renal apical transporters have led to a new concept of renal urate transport: urate-transporting multimolecular complex, or "urate transportsome," that may form an ultimate functional unit at the apical membrane of renal proximal tubules. Elucidation of urate transportsome will lead to the new drug development for hyperuricemia.     

Potassium transport across the frog retinal pigment epithelium

Previous experiments indicate that the apical membrane of the frog retinal pigment epithelium contains electrogenic Na:K pumps. In the present experiments net potassium and rubidium transport across the epithelium was measured as a function of extracellular potassium (rubidium) concentration, [K]0 ( [Rb]0). The net rate of retina-to-choroid 42K(86Rb) transport increased monotonically as [K]0 ( [Rb]0) increased from approximately 0.2 to 5 mM on both sides of the tissue or on the apical (neural retinal) side of the tissue. No further increase was observed when [K]0 ( [Rb]0) was elevated to 10 mM. Net sodium transport was also stimulated by elevating [K]0. The net K transport was completely inhibited by 10-4 M ouabain in the solution bathing the apical membrane. Ouabain inhibited the unidirectional K flux in the direction of net flux but had no effect on the back-flux in the choroid-to-retina direction. The magnitude of the ouabain-inhibitable 42K(86Rb) flux increased with [K]0 ( [Rb]0). These results show that the apical membrane Na:K pumps play an important role in the net active transport of potassium (rubidium) across the epithelium. The [K]0 changes that modulate potassium transport coincide with the light-induced [K]0 changes that occur in the extracellular space separating the photoreceptors and the apical membrane of the pigment epithelium.     

Potassium transport across the frog retinal pigment epithelium

Summary Previous experiments indicate that the apical membrane of the frog retinal pigment epithelium contains electrogenic NaK pumps. In the pressent experiments net potassium and rubidium transport across the epithelium was measured as a function of extracellular potassium (rubidium) concentration, [K] _o ([Rb] _o ). The net rate of retina-to-choroid⁴²K(⁸⁶Rb) transport increased monotonically as [K] _o ([Rb] _o ), increased from approximately 0.2 to 5mm on both sides of the tissue or on the apical (neural retinal) side of the tissue. No further increase was observed when [K] _o ([Rb] _o ) was elevated to 10mm. Net sodium transport was also stimulated by elevating [K] _o . The net K transport was completely inhibited by 10^–4 m ouabain in the solution bathing the apical membrane. Ouabain inhibited the unidirectional K flux in the direction of net flux but had not effect on the back-flux in the choroid-to-retina direction. The magnitude of the ouabain-inhibitable⁴²K(⁸⁶Rb) flux increased with [K] _o ([Rb] _o ). These results show that the apical membrane NaK pumps play an important role in the net active transport of potassium (rubidium) across the epithelium. The [K] _o changes that modulate potassium transport coincide with the light-induced [K] _o changes that occur in the extracellular space separating the photoreceptors and the apical membrane of the pigment epithelium.     

Potassium dependence of sodium transport in frog skin

22Na+ and 42K+ fluxes across the basolateral membrane of the isolated epithelium of frog skin were investigated with regard to dependence on K+ in the basolateral solution. When K+ was removed from the basolateral solution (K+-free Ringer), there was a transient rise in short circuit current (Isc) that could be eliminated by pretreatment with ouabain. Concurrently, the apparent sodium efflux across the basolateral membrane (JNa*13) showed either no change or an immediate (1-2 min) small decrease (approximately equal to 10%) that was followed by a small transient increase. K+ fluxes showed either no change or a small decrease under these conditions. JNa*13 was partially ouabain sensitive during all of the above treatments. Furosemide partially inhibited both sodium and potassium flux after K+-free treatment. The pump, as defined by ouabain sensitivity of Na+ flux, continued to work even after 20 minutes of K+-free treatment. Pump activity may be maintained by potassium leaking from the cells that is recycled by the pump. However, the ouabain-sensitive transient rise in Isc after K+-free treatment cannot readily be explained by changes in either Na+ or K+ flux. A change in pump coupling ratio provides one explanation for these data.     

A primary culture of distal convoluted tubules expressing functional thiazide-sensitive NaCl transport

Studying the molecular regulation of the thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC) is important for understanding how the kidney contributes to blood pressure regulation. Until now, a native mammalian cell model to investigate this transporter remained unknown. Our aim here is to establish, for the first time, a primary distal convoluted tubule (DCT) cell culture exhibiting transcellular thiazide-sensitive Na(+) transport. Because parvalbumin (PV) is primarily expressed in the DCT, where it colocalizes with NCC, kidneys from mice expressing enhanced green-fluorescent protein (eGFP) under the PV gene promoter (PV-eGFP-mice) were employed. The Complex Object Parametric Analyzer and Sorter (COPAS) was used to sort fluorescent PV-positive tubules from these kidneys, which were then seeded onto permeable supports. After 6 days, DCT cell monolayers developed transepithelial resistance values of 630 ± 33 Ω·cm(2). The monolayers also established opposing transcellular concentration gradients of Na(+) and K(+). Radioactive (22)Na(+) flux experiments showed a net apical-to-basolateral thiazide-sensitive Na(+) transport across the monolayers. Both hypotonic low-chloride medium and 1 μM angiotensin II increased this (22)Na(+) transport significantly by four times, which could be totally blocked by 100 μM hydrochlorothiazide. Angiotensin II-stimulated (22)Na(+) transport was also inhibited by 1 μM losartan. Furthermore, NCC present in the DCT monolayers was detected by immunoblot and immunocytochemistry studies. In conclusion, a murine primary DCT culture was established which expresses functional thiazide-sensitive Na(+)-Cl(-) transport.     

Modification of silver-enhanced sodium transport across toad skin

1. Silver stimulated short-circuit current and transepithelial potential difference. 2. Cysteine inhibited the silver-induced short-circuit current. 3. There was a dose-response inhibition of silver-induced short-circuit current by cysteine. 4. The silver-induced short-circuit current is carried by a net active sodium transfer from the outside to the inside bathing solution.     

Relationship between L-alanine and sodium ion transport in isolated renal tubules.

1. Rat renal tubules were isolated by incubation with collagenase. The Na+ concentration in the tubules at 37 degrees C was increased by additions of g-strophantin and L-alanine. The increase of Na+ in the presence of both g-strophantin and L-alanine was stronger than with either alone. 2. Radioactive sodium (22-Na), which was taken up by the tubules at 0 degrees C in K+-free medium, was more slowly washed out in the buffer with added g-strophantin than in the control buffer, but L-alanine had no effect. 3. At 0 degrees C incubation without K+, g-strophantin did not affect the 22-Na transport of the tubules. But under the same conditions, L-alanine increased Na+ uptake significantly, and in conjunction with it, L-alanine uptake was also increased. 4. The relationship between L-alanine uptake and intra- extracellular Na+ concentration gradients was linear. The ration of L-alanine to Na+ uptake at 0 degrees C was about 1:2. 5. In the incubation without K+ at 0 degrees C, L-alanine could be accumulated in tubules against the chemical concentration gradient (about 1.5-fold). 6. In the incubation without K+ at 37 degrees C, the L-alanine concentration in tubules after 5 min was already steady (Ci/Ce = 2.2), but with K+ it was not stabilized after 10 min. The ration Ci/Ce with K+ WAS HIGHER THAN WITHOUT K+. 7. G-Strophantin, p-hydroxymercuribenzoate, amiloride, and 2,4-dinitrophenol inhibited L-alanine uptake in the tubules and at the same time increased Na+ concentration. The relationship between the L-alanine uptakes inhibited by g-strophantin, amiloride and dinitrophenol, and the respective intra- extracellular Na+ concentration gradients was strikingly linear. But in the case of p-hydroxymercuribenzoate there was no correlation. 8. The results indicate that L-alanine transport into the renal tubules might be regulated mainly by the intra- extracellular Na+ concentration gradient and that inhibitors such as g-strophantin, amiloride, and dinitrophenol could have a secondary effect on the L-alanine transport which follows the change of Na+ concentration in cells. p-Hydroxymercuribenzoate might have an inhibiting effect on the binding of carrier with Na+ and/or L-alanine.     

Lipid methylation, sodium transport and the response to PTH in renal tubules

The possible role of progressive methylation of phosphatidylethanolamine to phosphatidylcholine on sodium transport was examined in a suspension of rabbit proximal convoluted tubules. The relation between progressive methylation and the action of parathyroid hormone on sodium transport in this nephron segment was also determined. Incubation of the suspended tubules for 10 minutes at 37 degrees C with 200 microM S-adenosyl-L-[3H]-methyl methionine, a methyl donor, revealed a significant rate of de-novo phosphatidylcholine synthesis. The inactive adenosine analogue, 3-deazaadenosine (DZA), had a significant inhibitory effect on lipid methylation. Despite the inhibition of methylation by DZA, the ouabain sensitive component of oxygen consumption, an index of sodium transport, was not affected. PTH significantly inhibited ouabain sensitive oxygen consumption but had no effect on the methylation of phosphatidylethanolamine. Inhibition of methylation by DZA, did not affect the inhibitory effect of PTH on oxygen consumption. These studies demonstrate that in the proximal convoluted tubule of the rabbit the progressive methylation pathway is present and that inhibition of this pathway does not affect sodium transport. In addition, these studies suggest that the inhibitory effect of PTH on sodium transport is not mediated by the methylation pathway.     

Potassium modulation of taurine transport across the frog retinal pigment epithelium

Net taurine transport across the frog retinal pigment epithelium-choroid was measured as a function of extracellular potassium concentration, [K+]o. The net rate of retina-to-choroid transport increased monotonically as [K+]o increased from 0.2 mM to 2 mM on the apical (neural retinal) side of the tissue. No further increase was observed when [k+]o was elevated to 5 mM. The [K+]o changes that modulate taurine transport approximate the light-induced [K+]o changes that occur in the extracellular space separating the photoreceptors and the apical membrane of the pigment epithelium. The taurine-potassium interaction was studied by using rubidium as a substitute for potassium and measuring active rubidium transport as a function of extracellular taurine concentration. An increase in apical taurine concentration, from 0.2 mM to 2 mM, produced a threefold increase in active rubidium transport, retina to choroid. Net taurine transport can also be altered by relatively large, 55 mM, changes in [Na+]o. Apical ouabain, 10(-4) M, inhibited active taurine, rubidium, and potassium transport; in the case of taurine, this inhibition is most likely due to a decrease in the sodium electrochemical gradient. In sum, these results suggest that the apical membrane contains a taurine, sodium co-transport mechanism whose rate is modulated, indirectly, through the sodium pump. This pump has previously been shown to be electrogenic and located on the apical membrane, and its rate is modulated, indirectly, by the taurine co-transport mechanism.     

Effects of carbon monoxide on ion transport across rat distal colon

The aim of the present study was to investigate whether carbon monoxide (CO) induces changes in ion transport across the distal colon of rats and to study the mechanisms involved. In Ussing chamber experiments, tricarbonyldichlororuthenium(II) dimer (CORM-2), a CO donor, evoked a concentration-dependent increase in short-circuit current (I(sc)). A maximal response was achieved at a concentration of 2.5·10(-4) mol/l. Repeated application of CORM-2 resulted in a pronounced desensitization of the tissue. Anion substitution experiments suggest that a secretion of Cl(-) and HCO(3)(-) underlie the CORM-2-induced current. Glibenclamide, a blocker of the apical cystic fibrosis transmembrane regulator channel, inhibited the I(sc) induced by the CO donor. Similarly, bumetanide, a blocker of the basolateral Na(+)-K(+)-2Cl(-) cotransporter, combined with 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid sodium salt, an inhibitor of the basolateral Cl(-)/HCO(3)(-) exchanger, inhibited the CORM-2-induced I(sc). Membrane permeabilization experiments indicated an activation of basolateral K(+) and apical Cl(-) channels by CORM-2. A partial inhibition by the neurotoxin, tetrodotoxin, suggests the involvement of secretomotor neurons in this response. In imaging experiments at fura-2-loaded colonic crypts, CORM-2 induced an increase of the cytosolic Ca(2+) concentration. This increase depended on the influx of extracellular Ca(2+), but not on the release of Ca(2+) from intracellular stores. Both enzymes for CO production, heme oxygenase I and II, are expressed in the colon as observed immunohistochemically and by RT-PCR. Consequently, endogenous CO might be a physiological modulator of colonic ion transport.     

Double dependence of organic acid active transport in proximal tubules of surviving frog kidney on sodium ions II. Relationship between counter-flows of fluorescein and sodium ion across cell layer

With the aid of a direct microfluorimetric method a dependence of organic onion (fluorescein) transport into proximal tubules of surviving frog kidney on Na⁺-flow in the opposite direction was studied. It was shown that the complete removal of Na⁺ from the tubules lumen resulted in inhibition of fluorescein transport of about 30%. After a specific inhibitor of sodium channels, amiloride (10^-3M) having been introduced into lumen of the tubules, the fluorescein transport was inhibited to the same extent. Amiloride affects only when Na⁺ is present in the tubular lumen. S present in the tubular lumen. Strophantin K (5 · 10^?5 M), a specific inhibitor of (Na⁺, K⁺)-ATPase, reduced fluorescein transport about twice. Substances increasing the 3′,5′-AMP level in cells (theophylline, NaF) and exogenous 3′,5′-AMP inhibited fluorescein transport while substance that decreased the 3′,5′-AMP level intracellularly (carbachol) stimulated it. For realization of these effects Na⁺ should be present in proximal tubules lumen.Thus, the various effects on the Na⁺ flow from lumen of the tubules to medium at the level of both the basal and apical membranes alter the rate of organic acid active transport from medium to lumen as a result of changes in the maximum rate of transport ($\text{V}$) with unchanged $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$. It is suggested that the system of Na⁺ extrusion from proximal tubules produces peritubular membrane-side (near the membrane) gradient of Na⁺ concentration which may be higher than the summary Na⁺ gradient between the medium and the cytoplasm. The magnitude of this gradient affects the maximal rate value of Na⁺-dependent organic acid transport. So, there is a double dependence of the active transport system on Na⁺, and the stages where Na⁺ is needed are: (1) the formation of a carrier-substrate-Na⁺ complex and (2) the production of substantial membrane-side Na⁺ gradient at the expense of Na⁺ extrusion from the tubules.     

Effect of 5-hydroxytryptamine on sodium transport across bullfrog skin

Summary 5-hydroxytryptamine, when present in the solution bathing the inside surface of bullfrog skin at concentrations of 0.25–25.0 mM, reduced both electrical potential difference and short-circuit current across the skin. The magnitude of reduction in potential difference and short-circuit current was dependent on 5 HT concentration. Reduction in sodium influx entirely accounted for the reduction in short-circuit current. Preliminary evidence suggested a competition between 5 HT and vasopressin in the production of their effects on sodium transport across the skin, while high Ca⁺⁺ concentrations and 5 HT seemed to act independently of each other.Dr. Henry C. and Bertha H. Buswell Fellow.     

Amphotericin B-induced sodium transport and water flow across rabbit corneal epithelium

We have determined fluid translocation across the cellular layers lining the cornea by measuring changes in corneal transparency. The loss of 1.3 μ1/cm² fluid from the stroma causes an increase of +1% in transparency. Amphotericin B ($\text{2 · 10}{}^{-6}\text{M}$) when added to the tear side (=mucosal side) of the epithelium causes a rapid increase in potential difference of $\text{12.3 ± 0.7}\text{mV}\text{(}\text{mean}\text{±}\text{S.E.}\text{, n=6}$) followed by a slower increase of $\text{18.6 ± 1.5}\text{mV}$. The electrical resistance is reduced from $\text{3.2 ± 0.3}\text{k}\text{Ω ·}\text{cm}{}^2\text{to}\text{0.6 ± 0.1}\text{k}\text{Ω ·}\text{cm}{}^2$. The resulting increase in calculated short circuit current is accompanied by a decrease in transparency at a rate of $\text{3.6 ± 1.0\%}\text{per h}$, corresponding to an uptake of fluid by the cornea of $\text{4.7 μ}\text{l}\text{·}\text{cm}{}^{-2}\text{·}\text{h}{}^{-1}$. Replacement of the fluid bathing the endothelial side of the cornea, in order to prevent water movement from the aqueous compartment into the stroma, did not significantly alter this uptake of fluid. Thus the epithelial fluid transport which is reported to be normally slightly secretory, becomes absorptive in the presence of amphotericin B. Serosal hypertonicity (20 mM mannitol) increases the water influx into the cornea induced by amphotericin B. These results indicate that amphotericin B induces sodium-selective channels in the epithelium leading to an accumulation of NaCl and water in the stromal layer of the cornea. Ouabain reduces the potential and calculated short circuit current in epithelia pretreated with amphotericin B. Following addition of ouabain, the NaCl and water accumulated in the stroma leak away resulting in a transient increase in transparency. Finally, a model is proposed that includes a stromal compartment involved in fluid transport and that agrees with the results presented here.