The Journal of Membrane Biology

Kone, Bruce; Brenner, Robert; Gullans, Steven

doi: 10.1007/BF01869600pmid: 2304068

The cellular mechanisms by which nephrotoxic heavy metals injure the proximal tubule are incompletely defined. We used extracellular electrodes to measure the early effects of heavy metals and other sulfhydryl reagents on net K+ and Ca2+ transport and respiration (QO2) of proximal tubule suspensions. Hg2+, Cu2+, and Au3+ (10−4 m) each caused a rapid net K+ efflux and a delayed inhibition of QO2. The Hg2+-induced net K+ release represented passive K+ transport and was not inhibited by barium, tetraethylammonium, or furosemide. Both Hg2+ and Ag+ promoted a net Ca2+ uptake that was nearly coincident with the onset of the net K+ efflux. A delayed inhibition of ouabainsensitive QO2 and nystatin-stimulated QO2, indicative of Na+, K+-ATPase inhibition, was observed after 30 sec of exposure to Hg2+. More prolonged treatment (2 min) of the tubules with Hg2+ resulted in a 40% reduction in the CCCP-uncoupled QO2, indicating delayed injury to the mitochondria. The net K+ efflux was mimicked by the sulfhydryl reagents pCMBS and N-ethylmaleimide (10−4 m) and prevented by dithiothreitol (DTT) or reduced glutathione (GSH) (10−4 m). In addition, both DTT and GSH immediately reversed the Ag+-induced net Ca2+ uptake. Thus, sulfhydryl-reactive heavy metals cause rapid, dramatic changes in the membrane ionic permeability of the proximal tubule before disrupting Na+, K+-ATPase activity or mitochondrial function. These alterations appear to be the result of an interaction of the metal ions with sulfhydryl groups of cell membrane proteins responsible for the modulation of cation permeability.

Flik, Gert; Schoenmakers, Theo; Groot, Jack; Os, Carel; Wendelaar Bonga, Sjoerd

doi: 10.1007/BF01869601pmid: 2137539

Measurements of unidirectional calcium fluxes in stripped intestinal epithelium of the tilapia,Oreochromis mossambicus, in the presence of ouabain or in the absence of sodium indicated that calcium absorption via the fish intestine is sodium dependent. Active Ca2+ transport mechanisms in the enterocyte plasma membrane were analyzed. The maximum capacity of the ATP-dependent Ca2+ pump (V m :0.63 nmol·min−1 mg−1,K m : 27nm Ca2+) is calculated to be 2.17 nmol·min−1·mg−1, correcting for 29% inside-out oriented vesicles in the membrane preparation. The maximum capacity of the Na+/Ca2+ exchanger with high affinity for Ca2+ (V m :7.2 nmol·min−1·mg−1,K m : 181nm Ca2+) is calculated to be 13.6 nmol·min−1·mg−1, correcting for 53% resealed vesicles and assuming symmetrical behavior of the Na+/Ca2+ exchanger. The high affinity for Ca2+ and the sixfold higher capacity of the exchanger compared to the ATPase suggest strongly that the Na+/Ca2+ exchanger will contribute substantially to Ca2+ extrusion in the fish enterocyte. Further evidence for an important contribution of Na+/Ca2+ exchange to Ca2+ extrusion was obtained from studies in which the simultaneous operation of ATP-and Na+-gradient-driven Ca2+ pumps in inside-out vesicles was evaluated. The fish enterocyte appears to present a model for a Ca2+ transporting cell, in which Na+/Ca2+ exchange activity with high affinity for Ca2+ extrudes Ca2+ from the cell.

Fisahn, Joachim; Lucas, William

doi: 10.1007/BF01869602pmid: 2304069

Reducing the pH of the bathing solution from 8.2 to pH 6 can induce an inversion of the extracellular current pattern that develops at the surface ofChara corallina internodal cells. A similar result can be obtained on some cells by changing the medium to a pH value of 10. In noninvertingChara cells the currents were strongly reduced when the pH value of the medium was changed between 3 and 11. Simultaneous measurements of theChara transmembrane potential in the acid and alkaline regions revealed that a light-induced electrical potential gradient of approximately 24 mV was present in the axial (or longitudinal) direction. Correlated to the external current pattern inversion was an inversion of this internal longitudinal voltage gradient. Reillumination ofNitella cells, after a period of darkness, often resulted in a complete inversion of the extracellular current pattern. These results are discussed in terms of spatial and temporal control of membrane transport processes, and in particular the control of current loops that pass through these cells.

Nielsen, Robert

doi: 10.1007/BF01869603pmid: 2304070

Prostaglandins are known to stimulate the active transepithelial Na+ uptake and the active secretion of Cl− from the glands of isolated frog skin. In the present work the effect of prostaglandin E2 (PGE2) on the glandular Na+ conductance was examined. In order to avoid interference from the Na+ uptake and the glandular Cl− secretion the experiments were carried out on skins where the Cl− secretion was inhibited (the skins were bathed in Cl− Ringer's solution in the presence of furosemide, or in NO 3 − Ringer's solution), and the active Na+ uptake was blocked by the addition of amiloride. Transepithelial current, water flow and ion fluxes were measured. A negative current was passed across the skins (the skins were clamped at −100 mV, basolateral solution was taken as reference). When PGE2, was added to the skins under these experimental conditions, the current became more negative; this was mainly due to an increase in the Na+ efflux. Together with the increase in Na+ efflux a significant increase of the water secretion was observed. The water secretion was coupled to the efflux of Na+, and when one Na+ was pulled from the basolateral to the apical solution via this pathway 230 molecules of water follwed. From the data presented it is suggested that this pathway for Na+ is confined to the exocrine glands.

Sugimoto, Tetsuo; Tanabe, Yasuto; Shigemoto, Ryuichi; Iwai, Masazumi; Takumi, Toru; Ohkubo, Hiroaki; Nakanishi, Shigetada

doi: 10.1007/BF01869604pmid: 2154581

We previously reported a novel rat membrane protein that exhibits a voltage-dependent potassium channel activity on the basis of molecular cloning combined with an electrophysiological assay. This protein, termedI sK protein, is small and different from the conventional potassium channel poroteins but induces selective permeation of potassium ions on its expression inXenopus oocytes. In this investigatiion, we examined cellular localization of ratI sK protein by preparing three different types of antibody that specifically reacts with a distinct part of ratI sK protein. Immunohistochemical analysis using these antibody preparations demonstrated that ratI sK protein is confined to the apical membrane portion of epithelial cells in the proximal tubule of the kidney, the submandibular duct and the uterine endometrium. The observed tissue distribution of ratI sK protein was consistent with that of theI sK protein mRNA determined by blot hybridization analysis. In epithelial cells the sodium, potassium-ATPase pump in the basolateral membrane generats a sodium gradient acrossthe epithelial cell and allows sodium ions to entere the cell through the apical membrane. Thus, taking into account the cellular localization of theI sK protein, together with its electrophysiological properties, we discussed a possible function of theI sK protein, namely that this protein is involved in potassium permeation in the apical membrane of epithelial cells through the depolarizing effect of sodium entry.

Bayliss, John; Reeves, W.; Andreoli, Thomas

doi: 10.1007/BF01869605pmid: 2304071

This paper provides the results of studies which characterized conductive36Cl− flux in basolaterally enriched membrane vesicles prepared from rabbit renal outer medulla. Conductive36Cl− uptake was studied under two different experimental conditions. In the first,36Cl− flux was driven by an inside positive voltage created with oppositely directed Cl− and gluconate gradients. In the second, an inwardly direct K+ gradient was used to drive36Cl− uptake. By these two methods, voltage-sensitive36Cl− uptake was shown to comprise about 45 and 65%, respectively, of the initial rates of total36Cl− flux. Separate paired studies demonstrated that the conductive36Cl− uptake was inhibited by the Cl− channel blocker diphenylamine-2-carboxylate (DPC) with an IC50 for DPC of 154 μm. The voltagedependent36Cl− uptake had an activation energy of 6.4 kcal/mole. This36Cl− conductance had an anion selectivity sequence of I−>Cl−≧NO 3 − ≫gluconate.

Reeves, W.; Andreoli, Thomas

doi: 10.1007/BF01869606pmid: 1689386

The present studies examined some of the properties of Cl− channels in renal outer medullary membrane vesicles incorporated into planar lipid bilayers. The predominant channel was anion selective having aP Cl/P K ratio of 10 and a unit conductance of 93 pS in symmetric 320mm KCl. In asymmetric KCl solutions, theI-V relations conformed to the Goldman-Hodgkin-Katz equation. Channel activity was voltage-dependent with a gating charge of unity. This voltage dependence of channel activity may account, at least in part, for the striking voltage dependence of the basolateral membrane Cl− conductance of isolated medullary thick ascending limb segments. The Cl− channels incorporated into the planar bilayers were asymmetrical: thetrans surface was sensitive to changes in ionized Ca2+ concentrations and insensitive to reducing KCl concentrations to 10mm, while thecis side was insensitive to changes in ionized Ca2+ concentrations, but was inactivated by reducing KCl concentrations to 50mm.

Alder, G.; Bashford, C.; Pasternak, C.

doi: 10.1007/BF01869607pmid: 2304072

Vero cells exposed to diphtheria toxin at pH 4.5 leak monovalent cations but not amino acids or phosphorylated metabolites; affected cells do not take up trypan blue. Monovalent cation leakage is inhibited by 1mmCd2+, but not by 1mmZn2+ or Ca2+. Cd2+ blocks calcein leakage from liposomes and closes diphtheria toxin-induced channels in lipid bilayers. It is concluded that translocation of the A fragment of diphtheria toxin across biological membranes does not depend on the formation of large stable pores, but that small Cd2+-sensitive pores may play a role.

Peracchia, Camillo

doi: 10.1007/BF01869608pmid: 2304073

Neutral-carrier pH-and Ca-sensitive microelectrodes were used to investigate the relationship between junctional electrical resistance and either pHi or [Ca2+]i in crayfish septate axons uncoupled by acidification. For measuring [Ca2+]i a new neutral carrier sensor sensitive to picomolar [Ca2+] and virtually insensitive to other ions was used. Uncoupling was induced by superfusing the axons with Na-acetate solutions (pH 6.3). With acetate, the time course of changes in junctional resistance differed markedly from that of pHi or [H+]i peaked 40–90 sec before junctional resistance. The difference in shape and peak time between pHi and junctional resistance curves caused significant hysteresis in the pHi versus junctional resistance relationship. In addition, junctional resistance maxima reached with slow acidification rates were 3–4 times greater than those with fast acidifications of similar magnitude. With acetate, [Ca2+]i, increased by approximately one order of magnitude from basal values of 0.1–0.3 μm. The curves describing the time course of changes in [Ca2+]i and junctional resistance matched well with each other in shape, peak time and magnitude. Both junctional resistance and [Ca2+]i recovered following a single exponential decay with a time constant of ∼2 min. Different rates of acidification caused increases in [Ca2+]i and junctional resistance comparable in magnitude. The data indicate that the increase in junctional resistance induced by acidification is more closely related to [Ca2+]i than to [H+]i.