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PubMed1975

A component of fluid absorption linked to passive ion flows in the superficial pars recta.

We studied salt and water absorption in isolated rabbit superficial proximal straight tubules perfused and bathed with solutions providing oppositely directed transepithelial anion gradients similar to those which might obtain in vivo. The perfusing solution contained 138.6 mM Cl- 3.8 mM HCO-3 (pH 6.6) while the bathing solution contained 113.6 mM Cl- and 25 mM HCO-3 (pH 7.4); the system was bubbled with 95% O2-5% CO2. At 37 degrees C, net volume absorption (Jv nl min-1 mm-1) was 0.32 +/- 0.03 (SEM); Ve, the transepithelial voltage (millivolts; lumen to bath), was +3.1 +/- 0.2. At 21 degrees C, Ve rose to +3.7 +/- 0.1 and Jv fell to 0.13 +/- 0.01 (significantly different from zero at P less than 0.001); in the presence of 10(-4)M ouabain at 37 degrees C, Ve rose to +3.8 +/- 0.1 and Jv fell to 0.16 +/- 0.01 (P less than 0.001 with respect to zero). In paired experiments, the ouabain- and temperature-insensitive moieties of Jv and Ve became zero when transepithelial anion concentration gradients were abolished. Titrametric determinations net chloride flux at 21 degrees C or at 37 degrees C with 10(-4) M ouabain showed that chloride was the sole anion in an isotonic absorbate. And, combined electrical and tracer flux data indicated that the tubular epithelium was approximately 18 times more permeable to Cl- than to HCO-3. We interpret these results to indicate that, in these tubules, NaCl absorption depends in part on transepithelial anion concentration gradients similar to those generated in vivo and in vitro by active Na+ absorption associated with absorption to anions other than chloride. A quantitative analysis of passive solute and solvent flows in lateral intercellular spaces indicated that fluid absorption occurred across junctional complexes when the osmolality of the lateral intercellular spaces was equal to or slightly less than that of the perfusing and bathing solutions; the driving force for volume flow under these conditions depended on the fact that sigmaHCO3 exceeded sigmaCl.

Absorption↗
PubMed1977

Enzymatic oxidation of isethionate to sulfoacetaldehyde in bacterial extract.

Isethionate degradation in a bacterial extract was shown by the isolation of enzymes and by identification of an intermediate to take place in two steps; dehydrogenation to sulfoacetaldehyde and desulfonation leading to the formation of sulfite and acetate. The enzyme responsible for isethionate oxidation in the presence of FAD was particulate in nature and a solubilized preparation obtained by extraction with buffer of low ionic strength had oxidizing activities against only isethionate and n-butanol among compounds tested. The enzyme was inhibited by thiol and carbonyl reagents.

Acetaldehyde↗
PubMed1993

Isolation of a glycosyl-phosphatidylinositol (GPI) from rat brain.

Brain lipids were labelled with [1-14C]-isethionyl acetimidate and purified by sequential thin layer chromatography. Four labelled peaks were obtained, the first ones migrating with the same Rf as glycosyl-phosphatidylinositol (GPI). Further proof of the isolation of GPI was obtained by the observations that 44.8% of the radioactivity associated with the lipid in peak I was converted to the water phase by the effect of a PI-specific phospholipase C, and that the soluble material so obtained produced a dose-dependent inhibition of cAMP-dependent protein kinase activity. These findings indicate a biological equivalence between GPI and its polar head group from rat brain and those described in other cell types, and are consistent with the proposed role of these molecules in cellular signalling.

Animals↗
PubMed1990

Asymmetric distribution of the phosphatidylinositol-linked phospho-oligosaccharide that mimics insulin action in the plasma membrane.

We have investigated the topography of a glycosyl-phosphatidylinositol implicated in insulin action by a combination of two complementary methods: (a) chemical labelling with a non-permeable (isethionyl acetimidate) and a permeable (ethyl acetimidate) probe; and (b) enzymatic modifications with beta-galactosidase (EC 3.2.1.23) or phosphatidylinositol-specific phospholipase C (EC 3.1.4.3). Using the first approach the majority of the glycosyl-phosphatidylinositol is found in the outer surface of intact hepatocytes, adipocytes, fibroblasts and lymphocytes, but not in erythrocytes which presented only a 20% of the total labelled glycosyl-phosphatidylinositol to the exterior. Upon insulin addition (10 nM), about 60% of the total glycosyl-phosphatidylinositol was hydrolysed in both hepatocytes and adipocytes but not in erythrocytes. In agreement with the extracellular localization in hepatocytes and with the proposed role of this glycolipid in insulin action, treatment of rat hepatocytes with beta-galactosidase from Escherichia coli, an enzyme that hydrolyses the oligosaccharide moiety of the glycosyl-phosphatidylinositol, cleaved 65% of the total glycophospholipid and blocked the effect of insulin (but not of glucagon) on pyruvate kinase (EC 2.7.1.40). Similar treatment with phosphatidylinositol-specific phospholipase C from Bacillus cereus hydrolysed 62% of the total glycosyl-phosphatidylinositol. From the various approaches used it is concluded that the majority of this glycophospholipid is at the outer surface in a variety of insulin-sensitive cells.

Animals↗
PubMed1983

Extracellular chloride replacement by isethionate induces abnormal spontaneous release of transmitter at the frog neuromuscular junction.

1 Replacement of chloride by isethionate in Ringer solution bathing frog skeletal muscle fibres induces, after a delay of about 30 min, marked mechanical activity which was blocked by tubocurarine. This effect is reversed by washing out the isethionate. 2 Miniature end plate potentials (m.e.p.ps) and giant potentials (potentials greater than or equal to 2 X modal value) were recorded intracellularly in normal Ringer and isethionate Ringer solution. 3 The frequency of m.e.p.ps was unaltered by isethionate. The proportion of giant potentials increased from 3% in normal Ringer to 24.5% in isethionate Ringer after 90 min. This effect is usually reversible if the exposure to isethionate does not exceed 2 h. 4 The giant potentials were large enough to initiate trains of action potentials and still occurred in the presence of tetrodotoxin or Ca2+-free Ringer. Isethionate produced no change in the tau D of miniature endplate currents. 5 Chloride replacement by propionate produced no change in the proportion of giant potentials. 6 It is suggested that the isethionate anion can induce giant potentials and the possible mechanism of action is discussed.

Alkanesulfonates↗
PubMed1999

Irritancy ranking of anionic detergents using one-time occlusive, repeated occlusive and repeated open tests.

Discrepancies between the one-time patch test and the wash test regarding the ranking of irritancy of detergents have been found in the literature. The aim of the present study was to investigate the concordance of irritancy rank order of 4 anionic detergents tested by 3 different exposure methods, namely one-time occlusive, repeated short-time occlusive and repeated short-time open tests. These detergents were sodium cocoyl isethionate (ISE), sodium lauryl sulfate (SLS), soap and disodium lauryl 3-ethoxysulfosuccinate (SUC). The reactions were evaluated by visual scoring and by transepidermal water loss (TEWL) measurement. When scored visually, the rank order in the one-time test was: SOAP > or = SLS > or = ISE > SUC. The other test methods yielded a different order: SLS > ISE > or = SOAP > SUC. A similar rank order was obtained with TEWL measurement for all exposure methods. Generally, the concordance among the different exposure methods was high when evaluated by TEWL. The concordance was lower when evaluation was performed by visual scoring. The present study demonstrates that the choice of exposure model and evaluation method may be important variables influencing the outcome of irritancy testing. It is proposed that the repeated open test is the best way to simulate most in-use situations where the uncovered skin is exposed to detergents. The repeated occlusive test or the one-time patch test may be better to simulate situations in which the skin is occluded after irritation by detergents.

Adolescent↗
PubMed1982

Exchange of isethionate between blood and tissues in adult and 7-day-old mice.

[14C] Isethionate was injected intramuscularly into adult and 7-day-old mice and the distribution of the label in the blood, brain, heart, liver and spleen was determined. Endogenous isethionate in these tissues was determined and approximate exchange rates calculated from the endogenous isethionate tissue levels and specific radioactivities after certain time intervals. The concentration of isethionate was higher and its exchange rates between plasma and tissues faster in the adult mice than in the 7-day-old mice. A close correlation between isethionate transport rates and tissue levels in vivo was obtained in both age groups. The tissues eliminated isethionate rapidly, even faster in the adult than in the 7-day-old mice. The elimination of isethionate showed three different components, fast, intermediate and slow, in the heart, liver and spleen of the adult mice, but only two components, fast and slow, in the other cases.

Alkanesulfonates↗
PubMed1975

Ion fluxes during the inhibitory junction potential in the guinea-pig taenia coli.

1. Contribution of different ions to the inhibitory junction potential (i.j.p.) in the guinea-pig taenia coli was studied by measuring the 42K, 24Na and 36Cl fluxes, the membrane resistance and the influence of various external ion concentrations. 2. The membrane resistance, as measured by the electrotonic potential, decreased transiently during the i.j.p. A maximal reduction of the electrotonic potential of about 50% was found at the top of the i.j.p. 3. The i.j.p. amplitude could be reduced by raising the external potassium concentration. Extrapolation of the relationship observed shows that the inhibitory response would be abolished at 115 mM potassium. Similar experiments were made in chloride-free medium, chloride being replaced by isethionate. Amplitude and time course of the response were not different in chloride containing Locke solution and chloride-free medium. 4. The half-times of 42K, 24Na and 36Cl effluxes during rest were 29, 10 and 9 min respectively. The 42K-efflux from the preparation was markedly increased to about three times the resting efflux during field stimulation. In low-chloride solution a similar effect on 42K-efflux was observed during field stimulation. Only a slight increase in the chloride efflux was observed but the sodium efflux was not affected during field stimulation. 5. From the results presented it is concluded that the inhibitory junction potential is caused by a selective increase in potassium permeability of the smooth-muscle cell membrane.

Animals↗
PubMed1980

Mouse pancreatic acinar cells: the anion selectivity of the acetylcholine-opened chloride pathway.

1. Anion replacement experiments were performed on superfused in vitro mouse pancreatic tissue and the effects on the electrical response of acinar cells to ACh investigated. 2. Electrical measurements were made with two micro-electrodes inserted into electrically coupled cells. ACh was applied by microionophoresis. Potential recordings were taken before, during and after changeover from the control superfusion fluid, containing Cl-, to one containing the substituted anion. 3. From the results obtained the tested anions were classified into three groups: I, Cl(-)-like anions: Br-, I- and NO3-, causing either no change or a negative displacement of the ACh null-potential, compared to that measured in the control Cl(-)-containing solution, and only small changes in the resting and stimulated electrical properties of the acinar cell, II, ions less permeable than Cl-: isethionate, acetate, sulphate and hippurate, showing a positive displacement of the ACh null-potential and a similar or increased resting cell input resistance, and III, methylsulphate and benzenesulphonate, causing a negatively displaced ACh null-potential but showing changes in the resting electrical properties of the acinar cells characteristic of anions in group II. 4. The ACh null-potential sequence, in order of decreasing negativity, was NO3- greater than or equal to benzenesulphonate greater than or equal to I- greater than or equal to methylsulphate greater than Br- greater than or equal to Cl- greater than isethionate greater than acetate greater than or equal to sulphate greater than hippurate. 5. Experiments involving the use of bicarbonate demonstrated that it does not contribute significantly to the value of the ACh null-potential. 6. The sequence of the anions in group I were compared to the Eisenman series I, suggesting that the ACh-opened Cl- pathway comprises a large hydrated ion channel bearing a lining of weak positive charges. 7. A quantitative relationship was sought between the ACh null-potential and extracellular Cl-. It was found that a tenfold reduction in the extracellular concentration resulted in a 15 mV positive shift of the null-potential.

Acetylcholine↗
PubMed1988

Evidence for co-transport of sodium, potassium and chloride in mouse pancreatic islets.

1. The presence of a loop diuretic-sensitive co-transport system for Na+, K+ and Cl- was tested in isolated pancreatic islets. 2. Substitution of Cl- with the impermeant anion isethionate or addition of frusemide both reduced the ouabain-resistant islets uptake of 86Rb+ (K+ marker) without affecting the ouabain-sensitive uptake or equilibrium content of 86Rb+. The effects of Cl- substitution and frusemide were overlapping. 3. D-Glucose reduced the ouabain-resistant islets uptake of 86Rb+. This effect was additive to the effect of Cl- substitution or frusemide. 4. Substitution of Cl- with isethionate or addition of frusemide both reduced the efflux of 86Rb+ from the islets. These effects were additive to the reduction of 86Rb+ efflux induced by D-glucose. 5. Substitution of K+ or Na+ with choline reduced the equilibrium content of 36Cl- in the pancreatic islets. 6. These data are compatible with the operation in the pancreatic beta-cells of a loop diuretic-sensitive co-transport system for Na+, K+ and Cl-, that may serve as an inwardly directed Cl- pump.

Animals↗
PubMed2000

Structural domains of chimeric dopamine-noradrenaline human transporters involved in the Na(+)- and Cl(-)-dependence of dopamine transport.

Catecholamine transporters constitute the biological targets for several important drugs, including antidepressants, cocaine, and related compounds. Some information exists about discrete domains of these transporters that are involved in substrate translocation and uptake blockade, but delineation of domains mediating the ionic dependence of the transport remains to be defined. In the present study, human neuronal transporters for dopamine and noradrenaline (hDAT and hNET) and a series of six functional chimeras were transiently expressed in LLC-PK1 cells. Substitution of Cl(-) by isethionate reveals that cassette IV (i.e., the region of the transporter encompassing transmembrane domain 9 through the COOH terminal) plays an important role in the Cl(-)- dependence of the uptake. Substitutions of Na(+) and NaCl by Tris(+) and sucrose, respectively, demonstrate that three different segments scattered across the transporter are involved in the Na(+)- dependence of the transport activity: cassette I (i.e., the region from the amino terminus through the first two transmembrane domains), cassette IV, and junction between transmembrane domains 3 to 5 and 6 to 8. Results of the present work also suggest that the use of Tris(+) as a substitute for Na(+) results in a biased estimate of the Hill number value for hDAT. This study provides useful clues for identifying specific residues involved in the uptake function of the catecholamine transporters.

Carrier Proteins↗
PubMed1983

Localization of the major dehydrogenases in two methylotrophs by radiochemical labeling.

The localization of prominent proteins in intact cells of two methylotrophic bacteria, Hyphomicrobium sp. strain X and bacterium W3A1, was investigated by radiochemical labeling with [14C]isethionyl acetimidate. In bacterium W3A1, trimethylamine dehydrogenase was not labeled by the reagent and is, therefore, an intracellular protein, whereas the periplasmic location of the methylamine and methanol dehydrogenases was evidenced by being readily labeled in intact cells. Similarly, an intracellular location of the trimethylamine and dimethylamine dehydrogenases in Hyphomicrobium sp. strain X was indicated, whereas methanol dehydrogenase was periplasmic.

Alcohol Oxidoreductases↗
PubMed1986

Chloride removal and excitation-contraction coupling in guinea pig ileal smooth muscle.

The effect of extracellular Cl (Cl-o) removal on contractions evoked by a selective muscarinic agonist, cis-2-methyl-4-dimethylaminomethyl 1,3-dioxolane methiodide (CD), and high K+ depolarizations in the isolated guinea pig ileal longitudinal muscle was studied. The replacement of Cl-o with impermeant anions, such as isethionate (Ise-), was found to selectively inhibit a portion of the initial phasic response to K+ and CD, leaving the secondary and sustained tonic responses unchanged. In Ca2+-free solutions, the loss of contractile responses to high K+ was faster and more pronounced in Cl--free compared with Cl--containing solutions. Furthermore, the uptake of Ca2+, as represented by 45Ca2+, from the saline solution was delayed and reduced in Ise--containing Cl-o-free solutions. Replacement of Cl-o with other impermeant anions, such as gluconate and methylsulphate, had a similar action on contractile activity as for Ise-replacement. Cl-o replacement with permeant anions, such as nitrate, however, did not significantly inhibit the phasic response and sometimes increased the tonic response to K+. These results indicate that there is a Cl-o-dependent Ca2+ pool in the guinea pig ileal longitudinal muscle and we speculate that this Cl-o-dependent Ca2+ pool is associated with membrane structures, such as calveolae, which would thus offer a degree of protection to depletion by removal of extracellular Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

Chloride and depolarization by acetylcholine in canine airway smooth muscle.

The role of chloride channels has been examined in canine tracheal smooth muscle by recording mechanical responses to field stimulation and to acetylcholine (ACh) and by sucrose gap recording of excitatory junction potentials and ACh-induced electrical changes. The results of substitution studies using isethionate for chloride provided evidence that a chloride conductance contributes to the resting potential. The extrapolated reversal potential for ACh-induced depolarization was positive to the resting potential. Isethionate substitution inhibited ACh-induced depolarization, consistent with a contribution from increased Cl- conductance to the depolarization induced by ACh. However, closure of K+ channels and opening of a non-specific cation channel could also contribute to depolarization. Further study of the effects of isethionate substitution during prolonged tissue exposure to chloride-free medium showed that retention or the accumulation of Ca2+ in intracellular stores was impaired. We conclude that effects of chloride deprivation on responses to ACh may reflect an early increase in Cl- conductance, but longer term changes reflect the requirement for this anion to maintain internal Ca2+ stores.

Acetylcholine↗
PubMed1989

Membrane effects of chloride substitutes in guinea pig gallbladder epithelial cells.

Differences in the responses of guinea pig gallbladder epithelial cells to replacement of luminal Cl- with either isethionate (I), gluconate (G), sulfate (S), or cyclamate (C) were investigated in vitro using intracellular microelectrode techniques. In prostaglandin E1 (PGE1)-treated tissues (10(-6) M, serosal side), where electrodiffusive apical membrane Cl- permeability (PCla) is high, replacement of luminal Cl- caused transient membrane depolarizations of similar magnitudes but different times to peak (C greater than G = S greater than I). The subsequent shifts in membrane voltages were, at steady state, straight correlated with the concomitant increases in apparent ratio of apical to basolateral membrane resistances (Ra/Rb). Increases followed the rank order I greater than G = S greater than C, which was also found to be the case in the peak membrane hyperpolarizations on restoring luminal Cl-. Under control conditions (no PGE1, low PCla), three of the substitutes caused a slow hyperpolarization, C greater than G = S, whereas an I-for-Cl- substitution evoked a transient depolarization and a drop in Ra/Rb. Under both control and PGE1 conditions, a transient depolarization followed luminal I-for-C substitution. Our results are best explained by a stimulatory effect of I (and, less marked, G and S) on PCla. Intrinsic effects of cyclamate are not ruled out; however, among the substitutes examined, it is the most inert.

Animals↗
PubMed1991

Electric properties of rat liver cell cultures on gas-permeable membranes.

In rat hepatocytes grown on gas-permeable membranes (Petzinger et al. In Vitro Cell. Dev. Biol. 24: 491-499, 1988), cellular and canalicular potentials as well as input resistances were measured using two-channel microelectrodes. In HCO3(-)-containing solutions, we found -30.9 +/- 0.4 (SE) (n = 141) and -13.9 +/- 1.4 mV (n = 22) for cell and canalicular membrane potentials, respectively. There was no dependence of these parameters on the age of the primary culture. Canalicular input resistance, however, increased from 13.3 +/- 2.0 M omega (n = 4) at day 1 after seeding to 36.1 +/- 5.0 M omega (n = 9) at day 2 and stabilized thereafter, while cell input resistance continuously decreased from 37.0 +/- 3.3 M omega at 1 h (n = 6) to 5.2 +/- 2.1 M omega (n = 27) at 3 days after preparation. In ion substitution experiments there were no changes in the transference numbers for K+, Na+, or Cl- that could account for this effect. Cable analysis, however, revealed that the decrease in input resistance reflects a time-dependent increase in electrical coupling between cells. We conclude that rat liver cells on gas-permeable membranes are highly suited for the quantitative analysis of cell-to-cell interaction. In addition, cells and canaliculi are readily accessible with two-channel microelectrodes, making this preparation a promising tool for electrophysiological analysis of hepatocellular transport mechanisms.

Animals↗