Role In Ion Transport Research Articles

Biomimetic polyesters and their role in ion transport across cell membranes.

Syntheses of biomimetic low-molecular weight poly-(R)-3-hydroxybutanoate mediated by three types of supramolecular catalysts are presented. The utility of these synthetic polyesters for preparation of artificial channels in phospholipid bilayers capable of sodium and calcium ion transport across cell membranes, is discussed. Further studies on possible applications of these bio-polymers for manufacturing drugs of prolonged activity are under way.

Cloning of porcine NRAMP1 and its induction by lipopolysaccharide, tumor necrosis factor alpha, and interleukin-1beta: role of CD14 and mitogen-activated protein kinases.

The gene for natural resistance-associated macrophage protein 1 (NRAMP1) plays a dominant role in controlling the resistance of inbred mice to infection with intracellular bacteria, such as Mycobacteria, Salmonella, and Leishmania. NRAMP1 is a membrane protein with a consensus transport motif present in one of the intracellular loops. Although its functions remain unclear, recent clues suggest that NRAMP1 protein plays a potential role in ion transport, which presumably accounts for the ability of this single protein to regulate the intraphagosomal replication of several species of antigenically unrelated intracellular pathogens. Expression of NRAMP1 in mice can be induced by lipopolysaccharide (LPS) or bacterial infection; however, little is known about the mechanisms of induction. Here, we report the cloning of the full-length cDNA for porcine NRAMP1, which had over 85% identity in amino acid sequence to its congeners from humans, mice, cattle, and sheep. As for its mammalian congeners, expression of porcine NRAMP1 mRNA was cell and tissue specific and was highest in macrophages. Investigation of the molecular mechanisms by which NRAMP1 is induced showed that LPS-induced expression in macrophages, neutrophils, and peripheral blood mononuclear cells was time and dose dependent and was mediated primarily through CD14. Induction of NRAMP1 required de novo protein synthesis, and mitogen-activated protein kinases (MAPK) were essential. Blockage of either p38 or p42/44 MAPK pathways suppressed the expression of NRAMP1 to basal levels. These findings suggest that bacterial infection and proinflammatory mediators induce NRAMP1 expression via activation of MAPK pathways.

Polyphosphate/poly-(R)-3-hydroxybutyrate) ion channels in cell membranes.

Among the most intriguing structures formed by inorganic polyphosphates (polyP) are their complexes with poly-(R)-3-hydroxybutyrates) (PHB) that form ion channels in lipid bilayers. All peptide and protein ion channels, as well as synthetic ion channels, are amphiphilic structures with an outer coat of non-polar residues and a lining of polar and charged residues (Urry 1985; Christensen et al. 1988; Nakano et al. 1990; Sansom 1991; Kobuke et al. 1992; Epand 1993; Fyles et al. 1993). These attributes are provided in a cooperative fashion by the two structurally distinct homopolymers, polyP and PHB. The polymeric anion, polyP, forms a ladder of cation binding sites that stretches across the bilayer, shielded from the hydrophobic environment by the amphiphilic solvating polyester, PHB. Complexes of polyP and PHB, located in the plasma membranes of diverse bacteria (Reusch and Sadoff 1983; Reusch et al. 1986, 1987), are the first nonproteinaceous, ion-selective channels discovered in biological cells. Their capacity to form voltage-activated, calcium-selective channels in planar lipid bilayers has been established (Reusch et al. 1995; Das et al. 1997). As yet, there is no direct evidence of their in vivo functions; however, a number of studies point to calcium involvement in important cellular functions in bacteria, such as chemotaxis (Matsushita et al. 1989; Tisa and Adler 1992) and cell division (Chang 1986; Smith 1995; Norris 1996). In addition, there is substantial evidence that polyP/PHB complexes may serve as calcium pumps and DNA channels. Here we will discuss the singular molecular characteristics of polyP and PHB that relate to their roles in ion transport, and consider how the two polymers act in synergy to form these interesting transmembrane ion channels.

An asparagine residue regulating conductance through P2X2 receptor/channels.

Single channel currents were recorded from Xenopus oocytes expressing wild-type and mutated P2X2 receptors. When 100 mM Na+ was used as the permeant cation, unitary currents of about 80 pS were recorded from the oocyte expressing the wild-type channels. The single channel conductance was roughly halved when Asn333 was replaced by Ile (N333I). A similar decrease in single channel currents was also observed when 100 mM Li+ or Cs+ was used as the permeant cation. With two other mutants, in which Asp315 was replaced by Val (D315V) or Tyr330 was replaced by lie (T333I), single channel conductance was almost the same as that of the wild-type channels. The results suggest that Asn333, which is believed to be involved in the channel pore, plays an essential role in ion transport through P2X2 receptor/channels.

Ionic mechanism and role of phytochrome-mediated membrane depolarisation in caulonemal side branch initial formation in the mossPhyscomitrella patens

In caulonemal filaments of the mossPhyscomitrella patens (Hedw.), red light triggers a phytochrome-mediated transient depolarisation of the plasma membrane and the formation of side branch initials. Three-electrode voltage clamp and ion flux measurements were employed to elucidate the ionic mechanism and physiological relevance of the red-light-induced changes in ion transport. Current-voltage analyses indicated that ion channels permeable to K+ and Ca2+ are activated at the peak of the depolarisation. Calcium influx evoked by red light coincided with the depolarisation in various conditions, suggesting the involvement of voltage-gated Ca2+ channels. Respective K+ fluxes showed a small initial influx followed by a dramatic transient efflux. A role of anion channels in the depolarising current is suggested by the finding that Cl− efflux was also increased after red light irradiation. In the presence of tetraethylammonium (10 mM) or niflumic acid (1 μM), which block the red-light-induced membrane depolarisation and ion fluxes, the red-light-promoted formation of side branch initials was also abolished. Lanthanum (100 μM), which inhibits K+ fluxes and part of the initial Ca2+ influx activated by red light, reduced the development of side branch initials in red light by 50%. The results suggest a causal link between the red-light-induced ion fluxes and the physiological response. The sequence of events underlying the red-light-triggered membrane potential transient and the role of ion transport in stimulus-response coupling are discussed in terms of a new model for ion-channel interaction at the plasma membrane during signalling.

Cell types in the esophageal epithelium of Anguilla anguilla (Pisces, Teleostei). Cytochemical and ultrastructural characteristics

The esophagus is an important osmoregulatory organ in euryhaline teleost fishes; however, its histochemical and ultrastructural characteristics have been studied in few species. The structure of the esophageal mucosa was analyzed in the European eel ( Anguilla anguilla L.) and compared to the findings reported for other species. The histological features of the esophageal mucosa, the histochemical characteristics of mucosecretory cells in the epithelial layer and the ultrastructural features of the different cell types in this layer in animals caught in fresh water and raised in the laboratory were analyzed. The role of mucus as a protective covering and its role in ion transport an the esophagus are discussed. In addition, comments on the role of tonofilament-rich epithelial cells in ion transport are made.

Immunolocalization of Na+, K+-ATPase in the gill epithelium of rainbow trout, Oncorhynchus mykiss.

The Na+,K+-ATPase (the sodium pump) plays a crucial role in ion transport in the fish gill. An immunocytochemical method has been optimized, using the mouse monoclonal antibody IgG alpha5, raised against the alpha subunit of the avian sodium pump, to localize Na+,K+-ATPase in fish gill cells. The method appears to be successful for the immunolocalization of Na+,K+-ATPase in both paraffin-embedded gill tissue sections and primary cultures of fish gill epithelial cells. The immunostaining has demonstrated that Na+,K+-ATPase-positive cells are mainly localized on the primary lamellae, in the interlamellar region, which is in agreement with the distribution of ion-transporting cells, also called chloride cells, as shown by electron microscopy. Na+,K+-ATPase-positive cells have been demonstrated for the first time in primary cultures of gill epithelial cells. Comparative labeling studies of primary cultures have shown that sites of Na+,K+-ATPase-positive cells correspond to sites of cells labeled with dimethylaminostyrylmethyl-pyridiniumiodine, a fluorescent mitochondrial probe for ion-transporting cells. The immunocytochemical detection method for Na+,K+-ATPase in cells is proposed as an easy and specific Na+-transport-related method to characterize and localize ion-transporting cells in primary cultures and in tissue sections of fish gill epithelium.

Contrasting roles in ion transport of two K(+)-channel types in root cells of Arabidopsis thaliana.

Plant roots accumulate K+ over a range of external concentrations. Root cells have evolved at least two parallel plasma-membrane K+ transporters which operate at millimolar and micromolar external [K+]: high-affinity K+ uptake is energised by symport with H+, while low-affinity uptake is assumed to occur via ion channels. To determine the role of ion channels in low-affinity K+ uptake, a characterisation of the principal K(+)-selective ion channels in the plasma membrane of Arabidopsis thaliana (L.) Heynh. cv. Columbia roots was undertaken. Two classes of K(+)-selective channels were frequently observed: one inward (IRC) and one outward (ORC) rectifying with unitary conductances of 5 pS, 20 pS (IRCs) and 15 pS (ORC), measured in symmetrical 10 mM KCl. The dominant IRC (5 pS) and ORC (15 pS) were highly cation-selective (PCl:PK < 0.025) but less selective amongst monovalent cations (PNa:PK approximately 0.17-0.3). Both the IRC and the ORC were blocked by Ba2+, Cs+ and tetra-ethyl-ammonium, whereas 4-aminopyridine and quinidine selectively inhibited the ORC. The ORC open probability was steeply voltage-dependent and ORC activation potentials were close to the potassium equilibrium potential (EK+), enabling ORCs to conduct mainly outward, but occasionally inward, K+ current. By contrast, gating of the 5-pS IRC was weakly voltage-ependent and IRC gating was invariably restricted to membrane potentials more negative than EK+, ensuring K+ transport was always inwardly directed.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of ramified electrochemical growth by a diffusive wave.

The role of ion transport during ramified growth in a dilute, binary electrolyte is investigated by obtaining a wave solution to effective equations derived by asymptotic analysis of the Nernst transport equations. The concentration profile exhibits a diffusion layer ahead of the growing tips, in agreement with experiment and theory. The non-Laplacian electric field is stronger in the diffusion layer than in the bulk, and cations are accelerated through the layer toward the tips. The well-known growth speed of the aggregate envelope, roughly equal to the speed of migrating anions in the bulk emerges as the characteristic wave speed of our equations. The ``copper ratio'' is also predicted and is linked to the regulation of growth by the diffusive wave. Finally, an estimate of the induction time for ramified growth is derived, based on the idea that a critical diffusion layer width must be attained.

The role of ion transport in the formation of sub-embryonic fluid by the embryo of the Japanese quail.

1. Using a modification of the method of New (1955) explanted blastoderms of the Japanese quail were cultured for up to 72 h, and the role of ions in the formation of sub-embryonic fluid (SEF) investigated. 2. Culture media deficient in either sodium or chloride ions reduced the volume of SEF secreted by up to a quarter. Ionic composition of the fluid was little altered, and sodium was transported against a concentration gradient. 3. Amiloride, an inhibitor of Na+/H+ exchange, reduced SEF production by half, whereas ionic composition and osmolality of the fluid was unchanged. 4. Likewise, acetazolamide, an inhibitor of carbonic anhydrase, reduced SEF production by the blastoderm, and left the ionic composition and osmolality of the fluid unaltered. 5. Neither furosemide or 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid at the concentrations used changed the volume of SEF formed or its ionic composition. 6. It is concluded that the secretion of SEF is dependent upon the active transport of sodium across the blastoderm; an amiloride-sensitive Na+/H+ exchanger and carbon dioxide hydration catalysed by carbonic anhydrase are also involved. 7. Furthermore, it is proposed that fluid transport across the blastoderm is the result of local osmotic gradients, not from a sub-embryonic fluid hyperosmotic to albumen, as has been suggested previously.

Immunolocalization of Na+, K(+)-ATPase, Ca(++)-ATPase, calcium-binding proteins, and carbonic anhydrase in the guinea pig inner ear.

The distribution of Na+, K(+)-ATPase, Ca(++)-ATPase, carbonic anhydrase, and calcium-binding proteins were investigated immunohistochemically in paraffin sections of guinea pig inner ears. Marginal cells of the stria vascularis, type II fibrocytes of the spiral ligament, and cells in supralimbal and suprastrial regions, were positive for Na+, K(+)-ATPase. Type I fibrocytes of the spiral ligament were positive for Ca(++)-ATPase, carbonic anhydrase, calmodulin and osteopontin. In the vestibular system, dark cells were positive for Na+, K(+)-ATPase. However, these cells and subepithelial fibrocytes were negative for Ca(++)-ATPase, carbonic anhydrase, and the calcium-binding proteins. In the endolymphatic sac, epithelial cells in intermediate and distal portions were positive for Na+, K(+)-ATPase, but the reaction was less than that in the stria. The same endolymphatic sac cells that were positive for Na+, K(+)-ATPase were also positive for Ca(++)-ATPase and calcium-binding proteins, but negative for carbonic anhydrase. The presence of Ca(++)-ATPase and calcium-binding proteins in the type I fibrocytes of the spiral ligament suggests that these cells are involved in mediating Ca++ regulation. Lower levels of Na+, K(+)-ATPase and the co-existence of Ca(++)-ATPase and calcium-binding proteins in the epithelial cells of the endolymphatic sac indicate that these cells have a distinctive role in ion transport that is different from that of the cells of the stria vascularis and vestibular dark cells.

Ion transport systems in membrane vesicles isolated from crustacean tissues

AbstractStudies with membrane vesicles isolated from crustacean gill have offered evidence for three major transport systems believed to play central roles in ion transport across the gill epithelium. First, ATP‐dependent sodium/potassium and sodium/ammonium exchanges have been demonstrated in Na+ + K+ ‐ATPase‐rich membrane preparations, supporting the concept that ammonium ion can serve as an effective counterion for sodium transport from cytosol to blood. Second, an amiloride‐sensitive Na+ /H+ antiporter, demonstrated using a pH‐sensitive fluorescent dye, has been shown to be electrogenic, apparently exchanging 2 sodium ions for 1 hydrogen ion. These two transport systems, the first in the basolateral membrane and the second likely in the apical membrane, can achieve net transport of sodium ions from water to blood. Third, a Cl−/HCO3− exchanger has been demonstrated in membrane vesicles from crab gill, most likely playing a central role in the sodium‐independent transfer of chloride across the epithelium. Finally, although no direct studies of calcium transport in crab gill have been reported, indirect evidence from other tissues suggests that part of the calcium flux across the gill may be mediated by Ca2+ /H+ exchange. © 1993 Wiley‐Liss, Inc.

Effects of intracellular ions on interleukin-1 beta production by lipopolysaccharide-activated human monocytes.

Following the observation that interleukin 1 beta (IL-1 beta) production in lipopolysaccharide (LPS)activated monocytes increases in concert with a rise in intracellular pH (pHi), the role of ion transport in IL-1 beta production was investigated. The amiloride analogue 5-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of the Na(+)-H+ antiporter, inhibited extracellular IL-1 beta. The replacement of Na+ in the culture medium with sucrose or choline chloride also prevented monocyte activation. The sodium ionophore monensin, in doses from 100 pM to 1 microM, potentiated LPS-stimulated extracellular IL-1 beta when compared with LPS alone. In the absence of LPS activation, monensin by itself at 10 nM stimulated IL-1 beta production to 63%. EIPA at 10 microM inhibited the Na+ influx, the rise in pHi, and intra- and extracellular IL-1 beta production in activated monocytes; this inhibition was reversed by 10 nM monensin. In the absence of bicarbonate, or in the presence of 10 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, the pHi of activated monocytes and the total protein synthesis did not change, but the production of IL-1 beta was inhibited. The data suggest that the stimulated influx of Na+ via the Na(+)-H+ antiporter regulates both pHi and IL-1 beta production in LPS-activated monocytes. The requirement for bicarbonate indicates an additional mechanism(s), separate from the modulation of pHi and intracellular Na+.

The role of ion transport in the electrochemical corrosion of fluoropolymers. Preparation and properties of n-doped polymeric carbon with mixed ion/electron conductivity

Electrochemical reductive corrosion of fluoropolymers by alkali metal amalgams leads to the formation of a compact uniform layer of reaction products on the fluoropolymer surface. This layer behaves as a solid electrolyte with mixed electronic-ionic conductivity; the electron/ion-conductive component in the layer is elemental carbon that is n-doped with alkali metal. The rate of the layer growth is controlled by transport of alkali metal cations, which increases substantially in the sequence K +<Na +<Li +.

Interactions between cutaneous ion-exchange mechanisms and acid–base balance in amphibians

It has been suspected for over 50 years that amphibian ion exchange involves independent transport of Na+ and Cl− in an inward direction across the skin in exchange for acidic cations and basic anions, respectively. Although a role for such exchange mechanisms has obvious utility in acid–base balance, their participation in this homeostatic process has only recently been documented. We now know that in aquatic Ambystoma tigrinum, the presence of NaCl in the water bathing the skin is required for acid–base regulatory responses to hypercapnia and exercise-induced lactacidosis. Acidotic and alkalotic conditions in the animals' extracellular fluid cause changes in both Na+ and Cl− influx and net flux which are consistent with a role for ion transport in acid–base balance. These processes appear to be under the control of both catecholamines and interrenal steroids.

Voltage-activated currents recorded from rabbit pigmented ciliary body epithelial cells in culture.

1. The whole-cell recording mode of the patch-clamp technique was used to investigate the presence of voltage-activated currents in the isolated pigmented cells from the rabbit ciliary body epithelium grown in culture. 2. In Ringer solution with composition similar to that of the rabbit aqueous humour, depolarizing voltage steps activated a transient inward current and a delayed outward current, while hyperpolarization elicited an inwardly rectified current. 3. The depolarization-activated inward current was mainly carried by Na+ and was blocked by submicromolar concentrations of tetrodotoxin. This current in many cells was sufficiently large to produce a regenerative Na+ spike. 4. The depolarization-activated outward current was carried by K+ and blocked by external TEA and Ba2+. Its activation appeared to be Ca2(+)-independent. 5. The hyperpolarization-activated inward current was almost exclusively carried by K+ and was blocked by Ba2+ and Cs+. For large hyperpolarizations below -120 mV, this current exhibited a biphasic activation with a fast transient peak followed by a slower sag, that appeared to be due to K+ depletion. 6. The voltage-dependent K+ conductances probably act to stabilize the cell membrane resting potential and may also play a role in ion transport. The function of the Na(+)-dependent inward current is unclear, but it may permit the electrically coupled epithelial cells of the ciliary body to conduct propagated action potentials.

The role of ion transport in the regulation of cell proliferation.

The stimulation of growth in a variety of cell types is followed by rapid changes in ion transport across the plasma membrane and in the intracellular concentration of various ions. The addition of various growth factors to fibroblasts, for example, causes stimulation of Na+ entry through the Na(+)-H+ antiport. This results in the alkalinization of the cytosol and an increase in intracellular Na+ concentration. The increased intracellular Na+ in turn stimulates the Na+/K+ pump, raising the concentration of K+ and lowering the Na+ toward normal. These changes in monovalent ion transport appear to be a necessary part of the proliferative response. In addition to the changes in cytosolic Na+, K+, and pH, a number of growth factors also cause a rapid increase in the cytosolic concentration of Ca2+. The additional Ca2+ appears to come from intracellular organelles, since the effect does not require Ca2+ in the extracellular medium. The change in intracellular Ca2+ concentration persists for only a few minutes. Changes in ion transport have been observed after the addition of mitogens to a variety of cell types, including epithelial cells. For example, we have found that stimulation of proliferation of MDCK (dog kidney epithelial) cells by either serum or vasopressin is followed by stimulation of the activity of the Na+/K+ pump. The manner in which these rapid changes in ion transport may play a role in signalling the onset of the mitogenic response will be discussed.

Musings on the role of ion transport on cell volume regulation.

Musings on the role of ion transport on cell volume regulation.

Ultrastructure of osmoregulatory organs in larvae of the brackish-water mosquito, Culiseta inornata (Williston).

The ultrastructure of the Malpighian tubules, ileum, rectum, anal canal, and anal papillae of larvae of the mosquito Culiseta inornata was examined. The Malpighian tubules, rectum, and anal papillae have many of the ultrastructural features characteristic of ion transport tissues, i.e., elaboration of the basal and apical membranes and a close association of these membranes with mitochondria. The Malpighian tubules possess two cell types, primary and stellate. The larval rectum of C. inornata is composed of a single segment containing a homogenous population of cells. In this respect, the larval rectum of C. inornata is distinct from that of saline-water species of Aedes. The cells in the larval rectum of C. inornata, however, closely resemble those of one cell type, the anterior rectal cells, of the saline-water mosquito Aedes campestris with regard to cell and nuclear size, the percentage of the cell occupied by apical folds, and mitochondrial density and distribution. No similarities can be found between the rectum of C. inornata and the posterior segment of the saline-water Aedes, which functions as a salt gland. On this basis, we have postulated that the rectum of C. inornata does not function as a site of hyperosmotic fluid secretion. The ultrastructure of the anal papillae of C. inornata is consistent with a role in ion transport. The significance of these findings to comparative aspects of osmoregulatory strategies in mosquito larvae is discussed.

Carbonic anhydrase histochemistry in rabbit and mouse kidneys

The presence of carbonic anhydrase activity in rabbit and mouse kidneys was examined using a histochemical procedure with plastic embedded sections stained by the modified version of the cobalt-phosphate method (Hansson, 1967, 1968; Ridderstrale, 1976). Proximal convoluted tubules (S1 and S2 segments) in both species were strongly positive for carbonic anhydrase activity on the membranes of the luminal, lateral, and basal surfaces. The apical cytoplasm beneath the brush border and the nuclei also stained positively for carbonic anhydrase. The S3 segment (pars recta) of the proximal tubule in the rabbit was positive on the luminal membrane, with somewhat less intensity seen on the lateral and basal surfaces. This segment in the mouse was completely negative. The first part of the thin limbs of long-looped nephrons exhibited strong staining in the mouse. Faint luminal staining was present on descending thin limbs of short-looped nephrons in the mouse. In the rabbit, both the medullary and cortical ascending thick segments of the limb of Henle were completely negative. In contrast, the medullary and cortical ascending thick limbs in the mouse kidney showed staining on all plasma membranes. The intercalated cells in the cortical and medullary portion of the collecting tubules stained positively for carbonic anhydrase in both species. The principal cells of the collecting duct in the cortex were negative in the rabbit and faintly positive in the mouse. The principal cells in the upper medullary collecting tubules in both species stained intensely along the luminal, lateral, and basal cell membranes. The papillary collecting ducts were largely negative in both the rabbit and the mouse. Some interstitial cells in the rabbit in the region of the papillary tip were strongly positive. We conclude that there is a marked difference in carbonic anhydrase activity within and between the renal tubular segments of the rabbit and the mouse. In addition, these distinct differences that exist between the two species correlated with known physiological roles in ion transport.