Potassium exchangeability, potassium permeability, and membrane potential: some observations in relation to protein synthesis in the early echinoderm embryo

In the sea urchin embryo the membrane potential undergoes a large hyperpolarization within 30–40 min after fertilization (from approximately ?10 to ?70 mV, inside negative). This is due to an increase in potassium permeability. Parallel to this is an increase in the exchangeability of internal K with the external medium, indicating an alteration in the internal compartmentalization of K. The ability of the embryos to incorporate labeled amino acid precursor into protein also increases during this period. It has been suggested that the membrane potential may regulate synthetic events within the cell. We have approached this question in the sea urchin embryo by investigating the relationships between membrane potential, membrane permeability, K compartmentalization, and protein synthesis. We have observed the following. Inhibition of the change in membrane potential by high K does not alter the ability of preloaded or pulse-labeled cells to incorporate labeled amino acid precursor. This suggests that the increase in membrane potential, per se, is not a direct regulatory factor for protein synthesis in these cells. Inhibition of the membrane potential change also does not block the decompartmentalization of intracellular K nor the increase in permeability to K. Furthermore, decompartmentalization of K is not an immediate event upon fertilization, and its gradual increase correlates in time with the increased ability to incorporate labeled precursor into protein.     

Constitutive eNOS-derived nitric oxide is a determinant of endothelial junctional integrity

Basal vascular endothelial permeability is normally kept low in part by the restrictiveness of interendothelial junctions (IEJs). We investigated the possible role of nitric oxide (NO) in controlling IEJ integrity and thereby regulating basal vascular permeability. We determined the permeability of continuous endothelia in multiple murine vascular beds, including lung vasculature, of wild-type mice, endothelial nitric oxide synthase (eNOS) null mice, and mice treated with NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME). Light and electron microscopic studies revealed that L-NAME treatment resulted in IEJs opening within a few minutes with a widespread response within 30 min. We observed a 35% increase in transendothelial transport of albumin, using as tracer dinitrophenylated albumin in mouse lungs and other organs studied. To rule out the involvement of blood cells in the mechanism of increased endothelial permeability, vascular beds were flushed free of blood, treated with L-NAME, and perfused with the tracer. The open IEJs observed in these studies indicated a direct role for NO in preserving the normal structure of endothelial junctions. We also used the electron-opaque tracer lanthanum chloride to assess vascular permeability. Lanthanum chloride was presented by perfusion to various vascular beds of mice lacking NO. Open IEJs were seen only in capillary and venular endothelial segments of mice lacking NO, and there was a concomitant increase in vascular permeability to the tracer. Together, these data demonstrate that constitutive eNOS-derived NO is a crucial determinant of IEJ integrity and thus serves to maintain the low basal permeability of continuous endothelia.     

Effect of various ionic compositions upon the membrane potentials during activation of sea urchin eggs

The membrane potentials of sea urchin (Hemicentrotus pulcherrimus) eggs before and after fertilization and their changes during the membrane elevation induced by intracellular electrical stimulation were recorded in solutions of various ionic compositions. Upon fertilization, the membrane potential (?10 mV) depolarized and reversed polarity by a few mV, then gradually returned to a new steady level ranging between ?50 and ?60 mV. The activation potential is closely associated with a transient increase in the membrane permeability. The potential of the unfertilized egg is hyperpolarized by monovalent anions (Br^?, Cl^? and NO₃^?) and depolarized slightly by K⁺. In contrast, the membrane of the fertilized egg is markedly depolarized by K⁺. Suppression of depolarization associated with an increase of the membrane permeability was recorded in Na-free medium (Tris-HCl). The selective increase in permeability to monovalent anions is thought to alternate with the selective increase in permeability to K⁺through the mediation of a transient increase of Na⁺-permeability at the time of fertilization. No causal relationship between the membrane elevation and the depolarization was established because the breakdown of the cortical granules occurs without depolarization or an increase in membrane permeability.     

Maturation of rat proximal tubule chloride permeability

We have previously shown that neonate rabbit tubules have a lower chloride permeability but comparable mannitol permeability compared with adult proximal tubules. The surprising finding of lower chloride permeability in neonate proximals compared with adults impacts net chloride transport in this segment, which reabsorbs 60% of the filtered chloride in adults. However, this maturational difference in chloride permeability may not be applicable to other species. The present in vitro microperfusion study directly examined the chloride and mannitol permeability using in vitro perfused rat proximal tubules during postnatal maturation. Whereas there was no maturational change in mannitol permeability, chloride permeability was 6.3 +/- 1.3 x 10(-5) cm/s in neonate rat proximal convoluted tubule and 16.1 +/- 2.3 x 10(-5) cm/s in adult rat proximal convoluted tubule (P < 0.01). There was also a maturational increase in chloride permeability in the rat proximal straight tubule (5.1 +/- 0.6 x 10(-5) cm/s vs. 9.3 +/- 0.6 x 10(-5) cm/s, P < 0.01). There was no maturational change in bicarbonate-to-chloride permeabilities (P(HCO3)/P(Cl)) in the rat proximal straight tubules (PST) and proximal convoluted tubules (PCT) or in the sodium-to-chloride permeability (P(Na)/P(Cl)) in the proximal straight tubule; however, there was a significant maturational decrease in proximal convoluted tubule P(Na)/P(Cl) with postnatal development (1.31 +/- 0.12 in neonates vs. 0.75 +/- 0.06 in adults, P < 0.001). There was no difference in the transepithelial resistance measured by current injection and cable analysis in the PCT, but there was a maturational decrease in the PST (7.2 +/- 0.8 vs. 4.6 +/- 0.1 ohms x cm2, P < 0.05). These studies demonstrate there are maturational changes in the rat paracellular pathway that impact net NaCl transport during development.     

Changes of uridine permeability during the maturation of tunicate eggs.

The time course of uridine uptake by eggs and embryos of the tunicate Ascidia callosa was studied using 5-min pulses of [³H]uridine at intervals from the unfertilized egg to the 16-cell embryo. The unfertilized egg is permeable to uridine, but 5 min after fertilization uptake begins to drop, reaching a minimum of 30% of the unfertilized rate about 30 min after fertilization. At 45 min after fertilization, permeability begins to increase, reaching a plateau about 3 hr after fertilization at the two-cell stage. The initial decrease in permeability occurs at first polar body production; the increase at 45 min is coincident with the formation of the second polar body. Substrate concentration experiments up to 200 μM show strict concentration dependence for uridine uptake. The inhibitors p-chloromercuribenzoate (PCMB), dinitrophenol (DNP), and thymidine have little, if any effect on permeability. Cold (?1°C) and Na⁺-free sea water inhibit uptake 60% during all three developmental stages. The changes in permeability may be indicative of temporary reorganization of the plasma membrane during the fertilization-initiated completion of meiosis.     

Functional Expression of p64, an Intracellular Chloride Channel Protein

p64 is a protein identified as a chloride channel by biochemical purification from kidney microsomes. We expressed p64 in HeLa cells using a recombinant vaccinia virus/T7 RNA polymerase driven system. Total cell membranes were prepared from infected/transfected cells and fused to a planar lipid bilayer. A novel chloride channel activity was found in cells expressing p64 and not in control cells. The p64-associated activity shows strong anion over cation selectivity. Single channels show prominent outward rectification with single channel conductance at positive potentials of 42 pS. The chloride channel activity is activated by treatment of the membranes with alkaline phosphatase and inhibited by DNDS and by TS-TM calix(4)arene. Whole membrane anion permeability was determined by a chloride efflux assay, revealing that membranes from cells expressing p64 showed a small but highly significant increase in chloride permeability, consistent with expression of a novel chloride channel activity. Received: 17 November 1997/Revised: 9 February 1998     

cAMP-regulated chloride currents in CHO cells.

We examined whether elevations in cAMP levels increase membrane chloride permeability in native CHO cells by measuring whole cell chloride currents and efflux of 125I and 36Cl. With 20 microM forskolin, no significant effect was seen on whole cell currents. However, 100 microM forskolin increased both whole cell chloride currents and the rate of 125I and 36Cl efflux. Forskolin-activated currents showed a linear current-voltage relationship in solutions with symmetrical chloride concentrations and reversal potential changed in the direction anticipated for a chloride-selective current when chloride was replaced with gluconate. These results indicate that native CHO cells exhibit cAMP-regulated chloride conductance pathways which become apparent only after large elevations in intracellular cAMP levels.     

Mechanism of thiyl radical-catalyzed isomerization of unsaturated fatty acid residues in homogeneous solution and in liposomes

Taurine is an important osmolyte involved in cell volume regulation. During regulatory volume decrease it is released via a volume-sensitive organic osmolyte/anion channel. Several molecules have been suggested as candidates for osmolyte release. In this study, we chose three of these, namely ClC-2, ClC-3 and ICln, because of their expression in rat astrocytes, a cell type which is known to release taurine under hypotonic stress, and their activation by hypotonic shock. As all three candidates were also suggested to be chloride channels, we investigated their permeability for both chloride and taurine under isotonic and hypotonic conditions using the Xenopus laevis oocyte expression system. We found a volume-sensitive increase of chloride permeability in ClC-2-expressing oocytes only. Yet, the taurine permeability was significantly increased under hypotonic conditions in oocytes expressing any of the tested candidates. Further experiments confirmed that the detected taurine efflux does not represent unspecific leakage. These results suggest that ClC-2, ClC-3 and ICln either participate in taurine transport themselves or upregulate an endogenous oocyte osmolyte channel. In either case, the taurine efflux of oocytes not being accompanied by an increased chloride flux suggests that taurine and chloride can be released via two separate pathways.     

Overexpression of human WNK1 increases paracellular chloride permeability and phosphorylation of claudin-4 in MDCKII cells

Pseudohypoaldosteronism type II (PHAII), an autosomal dominant disorder characterized by hypertension, hyperkalemia, and hyperchloremic acidosis, is reportedly due to mutations in WNK1 and WNK4 kinase genes. However, the pathogenesis of the disease remains unknown. Mutations in the WNK1 gene are the deletions in the first intron, which reportedly increases WNK1 mRNA expression. Thus, we generated WNK1 over-expressing stable cell lines using MDCKII cells to model the distal nephron of PHAII patients. Using these cell lines, we investigated whether increased WNK1 expression might affect paracellular chloride permeability and claudin phosphorylation, since we previously observed an increase in both with a disease-causing mutant WNK4. WNK1 expression in MDCKII cells increased chloride permeability two to threefold. Co-expression of wild-type WNK4 did not further increase WNK1-enhanced chloride permeability. WNK1 expression also induced phosphorylation of endogenous claudin-4 in MDCKII cells, as well as over-expressed claudin-4. Combined, these results suggest that increased WNK1 expression has the same effect on chloride permeability and claudin phosphorylation as the mutant WNK4. Thus, increased chloride shunt may be involved in the pathogenesis of PHAII caused by WNK1 mutations.     

A food dye, erythrosine B, increases membrane permeability to calcium and other ions

A widely used food additive erythrosine B, which has been implicated in minimal brain dysfunction in children was examined for its ability to increase membrane permeability to calcium ions. Planar phospholipid bilayer membranes become permeable to calcium, potassium and chloride ions and when erythrosine B is added to the aqueous phase at concentrations which were used by others to demonstrate effects on neuromuscular preparations. The observed increase in permeability to Ca2+ was of sufficient magnitude that equivalent effects on cells would seriously tax the systems which maintain low cytoplasmic Ca2+ levels. The permeability increase in the lipid bilayer membrane is time dependent and increases with erythrosine B concentration raised to a high power (4 to 7). This indicates that the permeability pathway is generated by the cooperative action of a number of erythrosine molecules. This permeability increases dramatically with increasing transmembrane voltage indicating that cells or organelles bearing potentials across their membranes should be particularly sensitive to the dye. We propose that the neurological effects of erythrosine stem from the increased Ca2+ permeability.     

Reduced expression and function of aquaporin-3 in mouse metaphase-II oocytes induced by controlled ovarian hyperstimulation were associated with subsequent low fertilization rate.

BACKGROUND/AIMS: Aquaporin-3 (AQP3), one isoform of water channel family, has been found to be expressed in mouse oocytes. The present study aimed to investigate whether functional AQP3 was expressed in oocytes induced by controlled ovarian hyperstimulation (COH), and whether altered oocyte AQP3 expression was associated with changes in fertilization rate. METHODS: Sixty ICR female mice were divided into two groups: COH and control. AQP3 mRNA expression of mouse metaphase II (MII) oocytes was quantified by real-time RT-PCR. The water permeability of oocytes was assessed with cell swelling test. The fertilization profiles of oocytes were generated via in vitro fertilization. RESULTS: AQP3 mRNA was expressed in both natural and COH-induced mouse oocytes. COH significantly reduced AQP3 mRNA expression. The volume of oocytes was significantly increased after exposure to hypotonic medium and pretreatment with HgCl(2) attenuated hypotonic medium-induced increase in oocyte volume and water permeability coefficient (Pf). Furthermore, the expression of AQP3, Pf and the fertilization rate were significantly lower in COH oocytes than those in control. CONCLUSION: AQP3 might play an important role in controlling oocyte quality and a low in vitro fertilization rate of COH mice might, in part, result from reduced AQP3 expression and water permeability in mouse oocytes.     

The localization of transport properties in the frog lens.

The selectivity of fiber-cell membranes and surface-cell membranes in the frog lens is examined using a combination of ion substitutions and impedance studies. We replace bath sodium and chloride, one at a time, with less permeant substitute ions and we increase bath potassium at the expense of sodium. We then record the time course and steady-state value of the intracellular potential. Once a new steady state has been reached, we perform a small signal-frequency-domain impedance study. The impedance study allows us to separately determine the values of inner fiber-cell membrane conductance and surface-cell membrane conductance. If a membrane is permeable to a particular ion, we presume that the conductance of that membrane will change with the concentration of the permeant ion. Thus, the impedance studies allow us to localize the site of permeability to inner or surface membranes. Similarly, the time course of the change in intracellular potential will be rapid if surface membranes are the site of permeation whereas it will be slow if the new solution has to diffuse into the intercellular space to cause voltage changes. Lastly, the value of steady-state voltage change provides an estimate of the lens' permeability, at least for chloride and potassium. The results for sodium are complex and not well understood. From the above studies we conclude: (a) surface membranes are dominated by potassium permeability; (b) inner fiber-cell membranes are permeable to sodium and chloride, in approximately equal amounts; and (c) inner fiber-cell membranes have a rather small permeability to potassium.     

Intracellular sodium activity in the sea urchin egg during fertilization

Fertilization of the sea urchin egg triggers a sequence of events that are necessary for metabolic derepression and stimulation of proliferation. Changes in intracellular Ca2+ and H+ activities regulate the sequence of events. Intracellular sodium activity is important in the regulation of the intracellular activities of these ions and may directly regulate metabolic events. Using Na+-sensitive microelectrodes we continuously measured the intracellular Na+ activity during fertilization. The results show an increase in intracellular sodium activity medicated by two pathways of Na+ entry: Na+ permeability increase during the fertilization potential and initiation of Na+-H+ exchange activity. Intracellular Na+ activity returned to unfertilized levels by 20 min after fertilization. This decrease was inhibited by ouabain, which suggests the activation of Na+, K+ ATPase during fertilization.     

Sequential biochemical and morphological events during assembly of the fertilization membrane of the sea urchin.

The fertilization membrane of Strongylocentrotus purpuratus undergoes changes in morphology, solubility, and permeability during the process of hardening. As the fertilization membrane elevates from the egg surface, it retains casts of the tips of the microvillous processes of the plasma membrane. The dome-shaped microvillar casts become angular at the same time that the fertilization membrane becomes resistant to solubilization in mercaptan solutions. 2-4 min after this morphological and chemical transition, the fertilization membrane becomes impermeable to the lectin conconavalin A, as monitored by binding of 125I- or fluorescein-labeled concanavalin A. Glycine ethyl ester inhibits the changes in morphology, solubility, and permeability, whereas sodium sulfite inhibits only the permeability block and resistance to solubilization by mercaptans. Parthenogenetic activation with the divalent ionophore, A23187, elicits fertilization membrane elevation more rapidly than does activation by fertilization; however, the morphological and permeability changes characteristic of hardening proceed more slowly. Elevation and hardening of the fertilization membrane thus appear to be discrete, multiple-step assembly processes that occur in fixed sequence, with kinetics that are affected by the mechanism of cortical granule exocytosis.     

Changes in osmotic water permeability of the eel gills during seawater and freshwater adaptation

Summary Changes in osmotic water permeability of the isolated gills of the Japanese eel,Anguilla japonica were studied during transfer to seawater or to fresh water. The water permeability increased gradually during the course of seawater transfer and attained a maximal level after 2 weeks. The water permeability of the freshwater eel gills was reduced when calcium ions were added to the incubation medium at a concentration of 1 mM, where-as no effect of the ion was observed on the gills of the seawater-adapted eel even at a higher concentration (10 mM). In contrast to seawater transfer, the water permeability decreased to a low level almost immediately (3 h) after transfer from seawater to fresh water. The acute reduction of the water permeability was also seen in the gills of the hypophysectomized eel after transfer to fresh water.The gradual increase in the gill water permeability during seawater transfer is correlated with an increase in the number of chloride cells. In scanning electron microscopy, chloride cells of seawater-adapted eel gills exhibit a pit-like structure, which was larger than in the freshwater eel. On transfer from seawater to fresh water, the pit diameter became smaller within 6 h. Hypophysectomy did not affect the change in gill surface structures during transfer to fresh water. The junctions between the chloride cells of seawater eel gills are reported to be of the leaky type. The parallel change in osmotic water permeability and in pit size of the chloride cells during seawater or freshwater transfer or after hypophysectomy suggests that these cells could provide a major route of water as well as ion movement.This paper is a portion of a thesis presented to Hokkaido University by t. Ogasawara in partial fulfilment of the requirements for Doctor of Fisheries     

Effect of claudins 6 and 9 on paracellular permeability in MDCK II cells

The neonatal proximal tubule has a lower permeability to chloride, higher resistance, and higher relative sodium-to-chloride permeability (P(Na)/P(Cl)) than the adult tubule, which may be due to maturational changes in the tight junction. Claudins are tight-junction proteins between epithelial cells that determine paracellular permeability characteristics of epithelia. We have previously described the presence of two claudin isoforms, claudins 6 and 9, in the neonatal proximal tubule and subsequent reduction of these claudins during postnatal maturation. The question is whether changes in claudin expression are related to changes in functional characteristics in the neonatal tubule. We transfected claudins 6 and 9 into Madin-Darby canine kidney II (MDCK II) cells and performed electrophysiological studies to determine the resultant changes in physiological characteristics of the cells. Expression of claudins 6 and 9 resulted in an increased transepithelial resistance, decreased chloride permeability, and decreased P(Na)/P(Cl) and P(HCO3)/P(Cl). These findings constitute the first characterization of the permeability characteristics of claudins 6 and 9 in a cell model and may explain why the neonatal proximal tubule has lower permeability to chloride and higher resistance than the adult proximal tubule.     

Effect of chloride withdrawal on the geometry of the T-tubules in amphibian and mammalian muscle

Summary The relative chloride permeabilities of the T-tubule membranes in mammalian (rat sternomastoid) and amphibian (toad sartorius) skeletal muscle fibers have been assessed from the change in volume of the T-tubules during chloride withdrawal from fibers exposed to low extracellular chloride concentrations. A 3.5- to 4.2-fold increase in T-tubule volume was found in mammalian fibers, and this was shown to be independent of the composition of the low chloride solution or the nature of the fixative used in preparation for electron microscopy. The increase in T-tubule volume was transient and was inhibited by factors which block chloride conductance, i.e., low pH, 2,4-dichlorophenoxyacetic acid, and nitrate ions. A small increase (1.48-fold) in T-tubule volume was seen in amphibian fibers when chloride ions were replaced by sulphate ions. No increase in volume was observed in amphibian T-tubules when methyl sulphate ions replaced chloride ions. The results support the idea that the chloride permeability of the T-tubule membrane is significantly higher in mammalian fibers than in amphibian fibers.     

Calcium-dependent events at fertilization of the frog egg: injection of a calcium buffer blocks ion channel opening, exocytosis, and formation of pronuclei

Eggs of Xenopus laevis were injected with a calcium buffer before insemination, to examine the effect of preventing or suppressing the sperm-induced increase in intracellular calcium on the fertilization potential, exocytosis, and pronuclear formation. Microinjection of BAPTA [(1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid)] at concentrations between 0.2 and 0.7 mM usually suppressed the fertilization potential to a series of transient depolarizations. The fertilization potential was completely inhibited when the final concentration of BAPTA in the egg was greater than 0.7 mM. These observations support the hypothesis that activation of the chloride conductance responsible for the fertilization potential depends on an increase in intracellular calcium. Exocytosis of cortical granules and elevation of the fertilization envelope were prevented by injecting BAPTA at concentrations greater than 0.2 mM. Injection of BAPTA to suppress the rise in calcium did not inhibit sperm entry and BAPTA-injected eggs were highly polyspermic. Examination by light and electron microscopy revealed that sperm decondensation and pronuclear formation were prevented by injection of the calcium buffer before insemination.     

The control of chick myoblast fusion by ion channels operated by prostaglandins and acetylcholine

Chick myoblast fusion in culture was investigated using prostanoid synthesis inhibitors to delay spontaneous fusion. During this delay myoblast fusion could be induced by prostaglandin E1 (PGE1), by raising extracellular potassium and by addition of carbachol. Carbachol-induced fusion, but not PGE-induced fusion, was prevented by the acetylcholine receptor blocker alpha-bungarotoxin. Fusion induced by any of these agents was prevented by the Ca channel blockers lanthanum and D600. The threshold for potassium-induced fusion was 7-8 mM; maximal fusion occurred at 16-20 mM. Low extracellular potassium inhibited spontaneous fusion. Intracellular potassium in fusion competent myoblasts was 101 m-moles/l cell. Calcium flux measurements demonstrated that high potassium increased calcium permeability in fusion-competent myoblasts. A 30-s exposure to high potassium or PGE1 was sufficient to initiate myoblast fusion. Anion-exchange inhibitors (SITS and DIDS) delayed spontaneous myoblast fusion and blocked fusion induced by PGE1 but not carbachol. Blocking the acetylcholine receptor shifted the dose-response relation for PGE-induced fusion to higher concentrations. PGE1-induced fusion required chloride ions; carbachol-induced fusion required sodium ions. Provided calcium channels were available, potassium always induced fusion. We conclude that myoblasts possess at least three, independent pathways, each of which can initiate myoblast fusion and that the PGE-activated pathway and the acetylcholine receptor-activated pathway act synergistically. We suggest that fusion competent myoblasts have a high resting membrane potential and that fusion is controlled by depolarization initiated directly (potassium), by an increase in permeability to chloride ions (PGE), or by activation of the acetylcholine receptor (carbachol); depolarization triggers a rise in calcium permeability. The consequent increase in intracellular calcium initiates myoblast fusion.     

Dog red blood cells: Na and K diffusion potentials with extracellular ATP

External ATP causes a prompt increase in the Na and K permeability of dog red blood cells. By manipulating intra- and extracellular ion composition it is possible to observe ATP-induced net fluxes which can be explained in terms of the contribution of Na or K diffusion potentials to the membrane potential. Measurements of membrane voltage by a fluorescent dye technique confirm the existence of such potentials. A rough calculation of chloride permeability gives a value of the order of 10(-8) cm/s, which agrees with results in other species. The cells appear to be somewhat more permeable to bromide than to chloride.