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PubMedDate not supplied

GABA uptake and release by a mammalian cell line stably expressing a cloned rat brain GABA transporter.

In order to facilitate study of the neuronal GABA transporter and provide a convenient system for potential drug screening, we have established a CHO cell line, designated 1F9, which stably expresses the cloned GABA transporter from rat brain (GAT-1). 1F9 cells transport GABA at levels approximately 300-fold higher than untransfected CHO cells, and GABA transport in these cells has the following properties: (1) a dependence on sodium and chloride ions; (2) higher sensitivity to neuronal subtype uptake inhibitors (DABA and ACHC) than to glial subtype inhibitors (beta-alanine and THPO); and (3) Km (2.5 microM) and IC50 values for various competitive ligands that are comparable with values determined in synaptosomes and brain slices. Given the fidelity with which the 1F9 cell line expresses these characteristics of the native neuronal GABA transporter, we have used it to further address GABA transporter activity. [3H]GABA uptake by 1F9 cells is inhibited approximately 50% by the chloride transport blockers DIDS and SITS. The GABA receptor agonists muscimol and baclofen also inhibit GABA transport; however, the receptor antagonists bicuculline and phaclofen have no effect. 1F9 cells also show release of [3H]GABA release is calcium independent, and is differentially affected by changes in the ion gradient, as well as by the presence of external substrates and uptake blockers. These experiments indicate that 1F9 cells provide a convenient system for the screening of GABA transport inhibitors.

Animals↗

PubMed1988

Cloned muscarinic receptor subtypes expressed in A9 L cells differ in their coupling to electrical responses.

The electrophysiological responses to cholinergic stimulation of four cloned muscarinic receptor subtypes (m1-m4) were studied in A9 L cells transfected with the expression plasmids of each of the different subtypes, using the tight-seal whole-cell recording technique. Cells transfected with m1 and m3 muscarinic receptor subtypes were hyperpolarized by acetylcholine (ACh), whereas m2- and m4-transfected cells did not respond to ACh concentrations of up to 1 mM. Stimulation of both m1 and m3 muscarinic receptor subtypes evoked outward currents in cells voltage-clamped at -50 mV, associated with an increase in membrane conductance. These outward currents were blocked by atropine but not by tubocurarine. The ACh-induced currents of m1- and m3-transfected cells primarily involved potassium ions, although chloride ions also contributed to a minor extent. The potassium and chloride conductances were blocked by barium or cobalt and by buffering the intracellular calcium to low levels with 5 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid, showing a dependence of these conductances on calcium. Thus, m1- and m3-transfected cells respond to ACh in a manner that is qualitatively similar, evoking calcium-dependent potassium and chloride conductances, whereas m2- and m4-transfected cells are not coupled to electrically detectable responses in A9 L cells.

Acetylcholine↗

PubMed1985

[Biological action and complex-forming properties of a macrotetrolide antibiotic complex].

Macrotetrolide antibiotic complexes containing 60-80 per cent of nonactin were studied and their high antibacterial activity was shown. The macrotetrolides had a disintegrating effect on the bacterial membrane, which was evident from induction of the protoplast lysis and release of low-molecular compounds from the bacterial cells. The complex-forming properties of nonactin with respect to the ammonium, potassium and sodium ions were investigated in vitro. The selectivity level with respect to the ammonium ions was higher. Nonactin incorporated into an artificial membrane provided a linear dependence of the delta mV on the ammonium chloride concentration within the ranges of 10(-1)-10(-5) M.

Anti-Bacterial Agents↗

PubMed2001

A new spectrophotometric method for the determination of total and ferric iron in rain water at the ppb level.

A new, simple, selective and sensitive spectrophotometric procedure for the on-site quantification of iron at nano-gram levels in atmospheric precipitations, i.e. rain as sample source is described. It is based on the color reaction of Fe3+ with SCN- ions in the presence of a cationic surfactant, i.e. cetylpyridinium chloride (CPC), in strong HCl solution, and subsequent extraction of the complex with N-octylacetamide into toluene or chloroform. The apparent molar absorptivity of the complex is 2.60 x 10(5) L mol(-1) cm(-1) at lambdamax = 480 nm at an enrichment factor (EF) of 10. The detection limit (causing higher absorbance than the sum of the blank absorbance (0.009) and 3 SD) is 5 ng mL(-1) Fe. Ions commonly associated with iron did not interfere in the present method. The effect of analytical variables, i.e. amount and type of the reagents, acidity, solvent, temperature, dilution, etc., in the determination of iron are discussed. The validity of the present method is checked with GF-AAS. The method has been applied to the determination of iron at the ppb level in rain water samples.

Journal Article↗

PubMed1996

Computational studies of ion-water flux coupling in the airway epithelium. II. Role of specific transport mechanisms.

Ion and water balance by the in vivo airway epithelium was investigated utilizing dynamic computer modeling. Parameters of the osmotically significant transport processes were varied to assess the sensitivity of water transport and fluid composition to transport perturbations. Establishment and regulation of water secretion represent a coordinated function of at least seven different ion transport processes: basolateral passive potassium transport, basolateral active sodium-potassium transport, basolateral sodium-potassium-chloride cotransport, apical passive sodium and chloride transport, and diffusion of sodium and chloride across the paracellular path. We found that apical chloride permeability at a level reported for cystic fibrosis is sufficient to cause the airway dehydration characteristic of cystic fibrosis. Given the reduction in apical chloride permeability in cystic fibrosis, a reduction in apical sodium permeability can potentially compensate completely for the airway dehydration associated with the cystic fibrosis genetic defect. Other simulations presented here address the importance of various membrane transport processes in airway epithelium water balance and the sensitivity of epithelium water balance to ion transport perturbations.

Animals↗

PubMed1979

Bicarbonate ATP-ase in ciliary body and a theory of Diamox effect on aqueous humor formation.

Bicarbonate was found to stimulate ATP breakdown by rabbit or cat ciliary body-iris homogenates. Maximum HCO3- stimulation of ATPase with Tris-Hepes buffer occured at pH 8.0. Acid pH and chloride ions in the media reduced the activity of the HCO3--stimulated ATPase. The Km for ATP was 0.55 mmolar and for HCO3-, 20 mmlar. HCO3- ATPase was not inhibited by acetazolamide added to in vitro. It is postulated that ATPase represents the linkage step of energy donor mechanism and active CT secretion in acid aqueous humors (human, cat.) or HCO3- secretion in alkaline aqueous humor (rabbit, guinea pig). Inhibition of Cl- or HCO3- secretion by acetazolamide results from decreased intracellular HCO3- levels which, in turn, reduces the stimulation of the HCO3- ATPase.

Acetazolamide↗

PubMed1998

Na-K-Cl cotransporter expression in the developing and senescent gerbil cochlea.

Changes in the cellular expression pattern of the Na-K-Cl cotransporter (NKCC) were investigated during postnatal development and with advancing age in the gerbil cochlea. At birth, faint immunostaining for NKCC was discernable in the developing stria vascularis (StV), Reissner's membrane, interdental cells and some relatively undifferentiated cells lining the cochlear partition. Between 2 and 4 days after birth (DAB) immunostaining persisted and increased in the future interdental, inner and outer sulcus and claudius cells but then disappeared from these sites by 8 DAB. In contrast, NKCC immunoreactivity in the StV increased progressively during development and approached adult levels by 12 DAB. Immunostaining for NKCC in subpopulations of fibrocytes in the inferior portion of the spiral ligament, the suprastrial region and the spiral limbus was first detectable between 10 and 12 DAB and staining intensity reached adult levels around 16 DAB. Changes in NKCC expression with advancing age generally mimicked those previously observed for Na,K-ATPase in focal regions of atrophic lateral wall. Diminished immunostaining was first seen in the StV, presumably associated with the involution of the marginal cell's basolateral processes. Further atrophy culminated in complete loss of immunostaining in the StV and an associated down-regulation of NKCC expression in spiral ligament transport fibrocytes. The marked similarities in the developmental and age-related expression patterns of NKCC and Na,K-ATPase point to a high level of functional cooperativity between these two ion transport mediators, which together provide an efficient mechanism for generating and maintaining high K+ levels in endolymph and the endocochlear potential.

Aging↗

PubMed2002

Novel CLCN1 mutations with unique clinical and electrophysiological consequences.

Myotonia is a condition characterized by impaired relaxation of muscle following sudden forceful contraction. We systematically screened all 23 exons of the CLCN1 gene in 88 unrelated patients with myotonia and identified mutations in 14 patients. Six novel mutations were discovered: five were missense (S132C, L283F, T310M, F428S and T550M) found in heterozygous patients, and one was a nonsense mutation (E193X) in a homozygous patient. While five patients had a clinical diagnosis of myotonia congenita, the patient with the F428S mutation exhibited symptoms characteristic of paramyotonia congenita--a condition usually thought to be caused by mutations in the sodium channel gene SCN4A. Nevertheless, no mutations in SCN4A were identified in this patient. The functional consequences of the novel CLCN1 sequence variants were explored by recording chloride currents from human embryonic kidney cells transiently expressing homo- or heterodimeric mutant channels. The five tested mutations caused distinct functional alterations of the homodimeric human muscle chloride ion channel hClC-1. S132C and T550M conferred novel hyperpolarization-induced gating steps, L283F and T310M caused a shift of the activation curve to more positive potentials and F428S reduced the expression level of hClC-1 channels. All showed a dominant-negative effect. For S132C, L283F, T310M and T550M, heterodimeric channels consisting of one wild-type (WT) and one mutant subunit exhibited a shifted activation curve at low intracellular [Cl(-)]. WT-F428S channels displayed properties similar to WT hClC-1, but expressed at significantly lower levels. The novel mutations exhibit a broad variety of functional defects that, by distinct mechanisms, cause a significant reduction of the resting chloride conductance in muscle of heterozygous patients. Our results provide novel insights into functional alterations and clinical symptoms caused by mutations in CLCN1.

Adult↗

PubMed2006

Smoking specifically induces metallothionein-2 isoform in human placenta at term.

Recently, we reported the presence of higher levels of metallothionein (MT) in placentas of smokers compared to non-smokers. In the present study, we designed experiments to separate and evaluate two isoforms of MT (MT-1 and MT-2) in placentas of smokers and non-smokers. Metallothionein was extracted and separated by ion-exchange high performance liquid chromatography (HPLC), previous saturation with cadmium chloride. Two peaks eluting at 6 and 12.5 min, corresponding to MT-1 and MT-2, respectively, were obtained. Metallothionein present in both peaks was identified by Western blot analysis using a monoclonal antibody directed against MT-1 and MT-2. Each isoform concentration was calculated after measuring its cadmium content by atomic absorption spectrometry with inductively coupled-plasma. In placentas of smokers, MT-2 levels increased by seven-fold compared to non-smokers, whereas MT-1 was not changed. Total placental cadmium and zinc concentrations, determined by atomic absorption spectrometry and neutron activation analysis, respectively, were higher in smokers. Metallothioneins levels were clearly in excess to bind all cadmium ions present in placentas. However, most of placental zinc remains unbound to MTs, although as much as twice zinc ions could be bound to MT in smokers. In conclusion, MT-2 is the main isoform induced by smoking, suggesting that this isoform could be involved in placental cadmium and zinc retention. This fact, which could contribute to reduce the transference of zinc to the fetus, may be associated to detrimental effects on fetal growth and development.

Blotting, Western↗

PubMed2006

The oxyhemoglobin dissociation curve in liver cirrhosis.

STUDY OBJECTIVES: To trace the entire oxyhemoglobin dissociation curve (ODC) in a cohort of cirrhotic patients in stable condition who were candidates for orthotopic liver transplantation (OLT). DESIGN: Prospective cohort study. SETTING: A large academic hospital. PATIENTS AND METHODS: We traced the entire ODC in whole blood in standard conditions (pH 7.4; PCO2, 40 mm Hg; temperature, 37 degrees C) for 50 cirrhotic candidates for OLT (27 men and 23 women) and 50 age- and height-matched healthy subjects (27 men and 23 women). All subjects were nonsmokers or ex-smokers for at least 5 years. We also measured 2,3 diphosphoglycerate (2,3 DPG) in RBCs, plasma ions, and arterial blood gases in all subjects according to standard methods. Mixed venous blood was also obtained from the 50 cirrhotic patients. RESULTS: Mean ODC was the same in the two groups. However, for the cirrhotic patients, the dispersion of the PO2 values of oxygen saturation percentage (SO2%) from 20 to 80% was significantly larger (p < 0.01 to p < 0.0001). In the cirrhotic patients, the mean PO2 for half-saturation of hemoglobin (P50) was 7.11 + 0.14 mEq/L chloride (p < 0.001) plus 0.36 mEq/L inorganic phosphate (p < 0.05) plus 0.25 micromol/gram of hemoglobin (gHb) 2,3 DPG (p < 0.00002) in absolute numerical values. Sodium, potassium, and calcium, three plasma ions disturbed in cirrhotic patients, did not contribute to determine the mean P50. DISCUSSION: In patients with cirrhosis, increased dispersion of PO2 values for a given level of SO2% may be related to four factors: (1) an observed alteration of the enzymes controlling the phosphoglycerate shunt; (2) hypothyroidism, which may affect 7 to 20% of patients with primary biliary cirrhosis; (3) the type of ongoing treatment, eg, diuretics and/or propranolol; and (4) plasma ion disturbances. CONCLUSIONS: We describe the ODC by three indexes: shape, position, and an index of dispersion of the PO2 values for a given level of SO2%. In addition, when the latter is increased, we suggest that other factors than pH, temperature, carbon dioxide, and inorganic phosphates are acting on the position of the ODC.

Chlorides↗

PubMed1999

Ion chromatography of nitrite at the ppb level with photometric measurement of iodine formed by post-column reaction of nitrite with iodide.

The difficulty in ion-chromatographic determination of nitrite in aqueous solutions containing a high concentration of chloride arises mainly from incomplete resolution of the peaks for these anions on the separation column whose efficiency is not high. A photometric measurement of iodine formed by a reaction of nitrite with iodide has been found to make it possible to determine, chromatographically, trace amounts of nitrite without any interference from chloride; chloride does not oxidize iodide to produce iodine. The proposed method was based on the separation of nitrite from matrix anions on a silica-based anion-exchange column with a 1.5.10(-3) M phthalate eluent (pH 5.0), followed by photometric measurement of the iodine (as triiodide) formed via a post-column reaction of the separated nitrite with iodide. The optimal conditions for the post-column reaction were established by varying the concentrations of iodide, copper(II) and nitric acid in a post-column-reaction solution and the length of a reaction tube. A calibration graph for nitrite, plotted as peak heights versus concentrations, was linear up to 1.50.10(-5) M (690 ppb). The detection limit, defined at S/N = 3, was 1.00.10(-7) M (4.60 ppb) nitrite. The presence of chloride ions up to 0.01 M did not give any interference to the determination of nitrite. This method was successfully applied to the determination of nitrite in lake water, river water, sewage works water and snow samples without any pretreatment.

Chromatography, Ion Exchange↗

PubMed1985

Evaluation of the electrophysiological consequences of GABA removal from the synaptic cleft by Na+ ion transport-coupled neuronal uptake.

The pre- and postsynaptic electrophysiological consequences of a carrier-mediated, Na+ ion transport-coupled removal of gamma-aminobutyric acid (GABA) from the relevant synaptic clefts are discussed. Assuming for the GABA internalization process a stoichiometry like GABAo + 3NA+o + K+i in equilibrium GABAi + 3Na+i + K+o and a synaptic cleft GABA maximal concentration of 100 microM we calculated the presynaptic depolarization associated with GABA removal between 11.5 and 38.2 mV. At the postsynaptic level the effect appears to be less marked.

Animals↗

PubMed1992

[Organization of the laboratory and practicability of methods for electrolyte determination].

Requirements, possibilities, and pitfalls of electrolyte (sodium, potassium, and chloride) analysis are reviewed within the light of the experiences in the Academic Hospital St. Radboud, Nijmegen, The Netherlands. In view of the ever increasing demands on short turnaround times, attention is paid to problems with specimen delivery, instrumentation and data distribution. The precision levels of available alternatives for electrolyte analysis namely: flame photometry, direct and indirect ion selective electrode methods, dry chemistry, and the newly developed enzymatic approach for sodium and potassium analysis are discussed.

Blood Gas Analysis↗

PubMed1991

Regulation of airway mucosal ion transport.

The secretion of fluid into the airway lumen is due in part to active chloride secretion across the epithelium. Changes in Cl secretion require coordinated changes in the turnover of several transport proteins in order to avoid lethal changes in cell volume. Several different Ca- and cAMP-dependent pathways effect this coordination. Many mediators raise cyclic AMP which activates protein kinase A which in turn opens the apical membrane Cl channel. Cyclic AMP also raises Cai (without change in levels of inositol triphosphate, IP3), and this Ca is probably responsible for activation of basolateral K channels and NaK2Cl cotransporters. Other mediators raise Cai by elevating IP3, and seem to stimulate chloride secretion entirely by Ca-dependent pathways with no change in cyclic AMP levels. Protein kinase C and arachidonic acid inhibit Cl secretion.

Animals↗

PubMed2001

Protein binding of glufosinate and factors affecting it revealed by an equilibrium dialysis technique.

We investigated the protein binding of glufosinate ammonium (GLF) and several factors affecting this binding using human serum albumin (HSA) and human volunteer serum under various conditions. The mean ratios of the free GLF (RFr-GLF) to 4% HSA were examined in the sera of patients described elsewhere at GLF levels from 1 microg/mL to 500 microg/mL; the range was found to be only from 0.80 to 0.88. Neither the incubation temperature nor buffers containing different chloride ion concentrations had any effect on the RFr-GLF to HSA. Moreover, the addition of heparin, glycoprotein-alpha1-acid (AAG), and sodium azide had no effect on the RFr-GLF. However, pH of the isotonic phosphate buffer and the addition of palmitic or oleic acid were seen to have an effect. In this study, the mean RFr-GLF to healthy human serum was 0.99. This high value was evidenced that GLF was rapidly excreted through the renal route.

Adult↗

PubMed1988

Properties of quisqualate-sensitive L-[3H]glutamate binding sites in rat brain as determined by quantitative autoradiography.

Quisqualate, a glutamate analogue, displaced L-[3H]glutamate binding in a biphasic manner, corresponding to "high-affinity" and "low-affinity" binding sites. High-affinity quisqualate sites were termed "quisqualate-sensitive L-[3H]glutamate" binding sites. Quisqualate-sensitive L-[3H]glutamate binding was regionally distributed, with the highest levels present in the cerebellar molecular layer. This binding was stimulated by millimolar concentrations of chloride and calcium. The stimulatory effects of calcium required the presence of chloride ions, whereas chloride's stimulatory effects did not require calcium. All of the L-[3H]glutamate binding stimulated by chloride/calcium was quisqualate sensitive and only weakly displaced by N-methyl-D-aspartate, L-aspartate, or kainate. At high concentrations (1 mM), the anion blockers 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid both reduced, by 41 and 43%, respectively, the stimulatory effects of chloride. At concentrations of 100 microM, kynurenate, L-aspartate, (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and L-2-amino-4-phosphonobutyric acid (L-APB) failed to displace quisqualate-sensitive L-[3H]glutamate binding in the cerebellar molecular layer. In the presence of KSCN, however, 100 microM AMPA displaced 44% of binding. Quisqualate-sensitive L-[3H]glutamate binding was not sensitive to freezing, and, in contrast to other chloride- and calcium-dependent L-[3H]glutamate binding sites that have been reported, quisqualate-sensitive binding observed by autoradiography was enhanced at 4 degrees C compared with 37 degrees C. Quisqualate-sensitive L-[3H]glutamate binding likely represents binding to the subclass of postsynaptic neuronal glutamate receptors known as quisqualate receptors, rather than binding to previously described APB receptors, chloride-driven sequestration into vesicles, or binding to astrocytic membrane binding sites.

Animals↗

PubMed1995

Role of CNP in human airways: cGMP-mediated stimulation of ciliary beat frequency.

Ciliated airway epithelial cells contribute to mucociliary transport systems via ciliary beating and electrolyte transport mechanisms. Both of these activities are regulated by agonists acting through intracellular calcium- and adenosine 3',5'-cyclic monophosphate (cAMP)-dependent processes (5, 15, 18, 27). This study examines the role of guanosine 3',5'-cyclic monophosphate (cGMP) in the regulation of both ciliary beat frequency (CBF) and electrolyte transport in human airway epithelia (HAE). In a previous report, cGMP production in HAE was observed after stimulation with either C-type natriuretic peptide (CNP) or sodium nitroprusside (SNP) (6). In this study, CNP was found to increase CBF by 30 +/- 6.9%, and this effect was mimicked by the cGMP analogue, 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP), but not by sodium nitroprusside. CNP-induced increases in CBF do not appear to be mediated by changes in either intracellular calcium or cAMP levels. Using modified Ussing chambers, we also investigated CNP's potential modulation of sodium and chloride transport rates. Neither CNP, nor SNP, nor 8-BrcGMP altered active ion transport rates. We conclude that CNP regulates ciliary beat via cGMP-dependent mechanisms, whereas no effect of CNP or cGMP on ion transport was detected.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1986

[Effect of various forms of inorganic nitrogen on the biosynthesis of rifamycin B].

To study the influence of various forms of inorganic nitrogen on biosynthesis of rifamycin B, ammonium sulfate, ammonium nitrate, ammonium chloride and sodium nitrate were compared. It was shown possible to use mineral salts containing nitrogen in reduced or oxidized form as fermentation medium components. Sodium nitrate induced early alkalization of the fermentation broth, while ammonium nitrate and ammonium chloride induced its early oxidation. Moreover, the chlorine ions inhibited the antibiotic biosynthesis. All this impaired normal development of the culture. Only ammonium sulfate provided the pH levels of the fermentation broth normal for the antibiotic biosynthesis and the highest levels of the antibiotic production. The positive role of this salt was due to the presence of both the ammonium group and the sulfogroup in the fermentation medium, since addition of the sulfogroup contained in the sulfates or sulfuric acid resulted only in partial increase of the antibiotic yield.

Culture Media↗