Catecholamine-stimulation of Cl− secretion in MDCK cell epithelium

Cultured epithelial monolayers of MDCK cells grown upon Millipore filter supports and mounted in Ussing chambers for transport studies respond to addition of $\text{5 · 10}{}^{\mathrm{?7}}\text{M}$ adrenalin from only the basal bathing solution by an increased short-circuit current, due both to an increased transmonolayer potential difference (basal solution electropositive) and an increased transmonolayer conductance. Measurement of tracer Na⁺, K⁺ and Cl^? fluxes demonstrate that the adrenalin-stimulated short-circuit current results primarily from basal to apical net Cl^? secretion. Half-maximal stimulation of the short-circuit current was observed at ($\text{3.1 ± 0.3) · 10}{}^{\mathrm{?8}}\text{M adrenalin}$; the order of potency of adrenergic agonists for short-circuit current stimulation was $\text{isoprenalin}\text{>}\text{adrenalin}\text{>}\text{noradrenalin}$, consistent with adrenalin action being mediated by a β-adrenergic receptor. The adrenalin-stimulated short-circuit current was sensitive to inhibition (75%) by basal additions of furosemide ($\text{1 · 10}{}^{\mathrm{?4}}\text{M}$); phloretin inhibition (54%, 57%) was observed from both epithelial surfaces. Amiloride (10^?4 M) and 4-acetamido-4-isothiocyanostilbene-2, 2′-disulphonic acid (SITS) (10 μM) were ineffective as inhibitors of the adrenalin response. The increased short-circuit current was sensitive to replacement of medium Na⁺ by choline (87%) and Tris (93%). Li⁺ was a partially effective substitute cation for Na⁺ · NO₃^?, and isethionate were ineffective substitutes for Cl^? whereas Br^? was partially effective. Partial replacement of medium Na⁺ by choline gave an upward-curving non-saturable dependence of the adrenalin-stimulated short-circuit current upon [Na]; partial replacement of Cl^? by NO₃^? in contrast gave a saturable increase with a $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of approx. 65 mM Cl^?.     

Energetics of coupled Na+ and Cl− entry into epithelial cells of bullfrog small intestine

Na⁺, K⁺ and Cl^? concentrations ($\text{c}{}_{\mathrm{j}}{}^{\mathrm{<mtext>i</mtext>}}$) and activities ($\text{a}{}_{\mathrm{j}}{}^{\mathrm{<mtext>i</mtext>}}$), and mucosal membrane potentials ($\text{E}{}_{\mathrm{<mtext>m</mtext>}}$) were measured in epithelial cells of isolated bullfrog (Rana catesbeiana) small intestine. Segments of intestine were stripped of their external muscle layers, and bathed (at 25°C and pH 7.2) in oxygenated Ringer solutions containing 105 mM Na⁺ and Cl^? and 5.4 mM K⁺. Na⁺ and K⁺ concentrations were determined by atomic absorption spectrometry and Cl^? concentrations by conductometric titration following extraction of the dried tissue with 0.1 M HNO₃. ¹⁴C-labelled inulin was used to determine extracellular volume. $\text{E}{}_{\mathrm{<mtext>m</mtext>}}$ was measured with conventional open tip microelectrodes, $\text{a}{}_{\mathrm{<mtext>Cl</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$ with solid-state Cl^?-selective silver microelectrodes and $\text{a}{}_{\mathrm{<mtext>Na</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$ and $\text{a}{}_{\mathrm{<mtext>K</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$ with Na⁺- and K⁺-selective liquid ion-exchanger microelectrodes. The average $\text{E}{}_{\mathrm{<mtext>m</mtext>}}$ recorded was ?34 mV. $\text{c}{}_{\mathrm{<mtext>Na</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$, $\text{c}{}_{\mathrm{<mtext>K</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$ and $\text{c}{}_{\mathrm{<mtext>Cl</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$ were 51, 105 and 52 mM. The corresponding values for $\text{a}{}_{\mathrm{<mtext>Na</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$, $\text{a}{}_{\mathrm{<mtext>K</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$ and $\text{a}{}_{\mathrm{<mtext>Cl</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$ were 18, 80 and 33 mM. These results suggest that a large fraction of the cytoplasmic Na⁺ is ‘bound’ or sequestered in an osmotically inactive form, that all, or virtually all the cytoplasmic K⁺ behaves as if in free solution, and that there is probably some binding of cytoplasmic Cl^?. $\text{a}{}_{\mathrm{<mtext>Cl</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$ significantly exceeds the level corresponding to electrochemical equilibrium across the mucosal and baso-lateral cell membranes. Earlier studies showed that coupled mucosal entry of Na⁺ and Cl^? is implicated in intracellular Cl^? accumulation in this tissue. This study permitted estimation of the steady-state transapical Na⁺ and Cl^? electrochemical potential differences (Δμ&#x0304;Na and Δμ&#x0304;Cl). Δμ&#x0304;Na (?7000 J · mol^?1; cell minus mucosal medium) was energetically more than sufficient to account for Δμ&#x0304;Cl (1000–2000 J · mol^?1).     

On the interrelations of the Na+-and Cl?-influxes in the pulmonate freshwater snailLymnaea stagnalis

Summary In the freshwater snailLymnaea stagnalis the influxes of Na⁺ and Cl^– were studied at different external concentrations of these ions. The characteristies of the Na⁺- and Cl^–-influxes are similar with respect to saturation kinetics,K _m (0.1 mM) and activation by low-salt adaptation. In short-term experiments the Na⁺- and Cl^–-influxes are independent. Because of the counter-ions (H⁺ and HCO ₃ ^– ) involved, this indicates a potential acid-base regulatory capacity. Low-salt adaptation, due to either Na⁺-or Cl^–-depletion, activates both the Na⁺- and the Cl^–-influx. It is suggested that under both conditions the number of active integumental pumps, involved in Na⁺- as well as in Cl^–-uptake, is increased.     

Modification of the Cl−-activated arginine aminopeptidases from rat liver and human erythrocytes: A comparative study

The amino acid residues important for the catalytic activity of the Cl^?-activated arginine aminopeptidases from human erythrocytes and rat liver were studied using enzyme modification. The general inhibition characteristics were similar with both enzymes. Inactivation with 5,5′-dithiobis-(2-nitrobenzoic acid) revealed one essential SH-group per active enzyme unit in both aminopeptidases. l-Arginyl-l-phenylalanine and N-l-arginyl-2-naphthylamide protected the enzymes against inactivation by DTNB, the former substrate being more effective. The rat liver enzyme was more sensitive to DTNB than the erythrocyte enzyme. Titration with DTNB revealed only fast reacting SH-groups in rat liver APB (mean 7.8). The erythrocyte enzyme, however, revealed SH-groups which reacted fast with low concentrations of DTNB, while high concentrations of DTNB or SDS treatment were needed to reveal all enzyme SH-groups (mean 8.0). The presence of at least one essential imidazole group in the erythrocyte enzyme was indicated by photooxidation in the presence of methylene blue, as previously found with the rat liver enzyme (5., 22.). The pH dependence curves of both enzymes also supported the presence of SH- and imidazole groups at or near the active site. Thus, the functional groups identified were the same for both enzymes. Neither enzyme had essential COOH or arginyl groups and they did not contain any zinc. The absence of Zn suggests that the reaction mechanism recently presented by other authors, based on the presence of Zn in the active center, does not apply to the Cl^?-activated arginine aminopeptidases. Accordingly, this enzyme group cannot be classified to metallopeptidases.     

Electrically silent cotransport of Na+, K+ and Cl− in ehrlich cells

A cotransport system for Na⁺, K⁺ and Cl^? in Ehrlich cells is described. It is insensitive towards ouabain but specifically inhibited by furosemide and other ‘high ceiling’ diuretics at concentrations which do not affect other pathways of the ions concerned. As the furosemide-sensitive fluxes of these ions are not affected by changes in membrane potential, and as their complete inhibition by furosemide does not appreciably alter the membrane potential, they appear to be electrically silent. Application of the pulse-response methods in terms of irreversible thermodynamics reveals tight coupling between the furosemide-sensitive flows of Na⁺, K⁺ and Cl^? ($\text{q}$ close to unity for all three combinations) at a stoichiometry of 1 : 1 : 2. The site for each of the ions appears to be rather specific: K⁺ can be replaced by Rb⁺ but not by other cations tested whereas Cl^? can be poorly replaced by Br^? but not by NO₃^?, in contradistinction to the Cl^?-OH^? exchange system. The cotransport system appears to function in cell volume regulation as it tends to make the cell swell, thus counteracting the shrinking effect of the ouabain-sensitive (Na⁺, K⁺) pump.The experiments presented could not clarify whether the cotransport process is a primary or secondary active one; while incongruence between transport and conjugated driving force seems to indicate primary active transport, it is very unlikely that hydrolysis of ATP supplies energy for the transport process, since there is no stimulation of ATP turnover observable under operation of the cotransport system.     

A comparative study of the mechanism of Na+ and Cl? excretion by the gill ofMugil capito andFundulus heteroclitus: Effects of Stress

Summary In seawater (SW)-adaptedMugil andFundulus, gill effluxes of Na⁺ and of Cl^– and the simultaneously recorded transgill potential (P.D.) differ according to whether they are measured in stressed or rested animals.In rested animals of the two species, transfer to Ringer's solution considerably reduces the P.D. but not . InFundulus, is also decreased. Transfer of the two species from SW to fresh water (FW) reduces and by 75 to 85% and leads to a large inversion of P.D. When K⁺ is added to FW, a gill depolarization occurs, as well as a large increase of and .These results suggest that: 1) the P.D. originates primarily from the diffusion of cations, the gill permeability to Na⁺ ( ) being greater than that to Cl^– ( ), 2) a Cl^–/Cl^– exchange independent of P.D. is associated with the Cl^– pump; 3) Cl^– pump activity is linked to Na⁺/K⁺ exchange which in turn is associated to a Na⁺/Na⁺ exchange diffusion mechanism.In stressed individuals of the two species, the P.D. in SW, as well as the P.D. changes observed during transfer experiments, are considerably reduced. The decrease of and observed after transfer from SW to FW are also minimised. Changes are smaller inFundulus. The decrease of P.D. characterizing stressed animals may be at least in part due to a 3 to 4 fold increase of which becomes equal to in both species.As a result of stress, the K⁺-activated Na⁺ and Cl^– excretion mechanisms are totally inhibited inFundulus and partially so inMugil.Stress response seems more intense inFundulus and recovery from stress faster inMugil.     

Potential difference responses due to K+, Na+ and Cl− changes in Bullfrog antrum with and without HCO3−

The effect of changing [K⁺], [Na⁺] and [Cl^?] in nutrient solution was studied in bullfrog antrum with and without HCO₃^? in nutrient. In 25 mM HCO₃^? (95% O₂/5% CO₂) and in zero HCO₃^? (100% O₂), nutrient pH was maintained at 7.3. Changing from 4 to 40 mM K⁺ or from 81 to 8.1 mM Cl^? gave a decrease 10 min later in transmucosal PD (nutrient became more negative) — a normal response. These responses were less in zero than in 25 mM HCO₃^?. A decrease from 102 to 8 mM Na⁺ decreased PD (anomalous response of electrogenic NaCl symport). This effect was attenuated or eliminated in zero HCO₃^?. In contrast, change from 4 to 40 mM K⁺ gave initial anomalous PD response and change from 102 to 8 mM Na⁺, initial normal PD response with either zero or 25 mM HCO₃^?. Both responses were associated with (Na⁺ + K⁺)-ATPase pump and were greater in zero than in 25 mM HCO₃^?. Initial PD increases in zero HCO₃^? are explained as due to increase in the resistance of passive conductance and/or NaCl symport pathways. Thus, removal of HCO₃^? modifies conductance pathways of nutrient membrane.     

Effects of cytochalasin B on water, Na+ and Cl? exchanges in the gill of seawater adapted mulletMugil capito

Summary Electron microscopy study shows that cytochalasin treatment of the mullet damages the microfilaments system in the apex of gill ionocytes: the microfilaments are reduced in number and shortened. Cytochalasin causes a reduction of transgill potential difference and an increase of the Na⁺ and Cl^– blood concentration, of the diffusional water permeability of the gill, of the Na⁺ branchial influx and of Cl^– efflux. The increase of the Na⁺ influx may result in a reduction of the Na⁺ net excretion flux compared to the control. The increased permeability in cytochalasin treated fish facilitates the Cl^– entry probably leading to a reduction of the net Cl^– excretion. The partial inhibition of the K⁺ dependent components of Na⁺ and Cl^– effluxes also contributes to the reduction of Na⁺ and Cl^– excretion. The role of microfilaments in the mechanisms of ionic excretion by the gill is discussed.     

Secretion of HCO 3 − /OH− in cortical distal tubule of the rat

Secretion of bicarbonate has been described for distal nephron epithelium and attributed to apical Cl^–/HCO ₃ ^– exchange in beta-intercalated cells. We investigated the presence of this mechanism in cortical distal tubules by perfusing these segments with acid (pH 6) 10 mm phosphate Ringer. The kinetics of luminal alkalinization was studied in stationary microperfusion experiments by double-barreled pH (ion-exchange resin)/1 m KCl reference microelectrodes. Luminal alkalinization may be due to influx (into the lumen) of HCO ₃ ^– or OH^–, or efflux of H⁺. The magnitude of the Cl^–/ HCO ₃ ^– exchange component was measured by perfusing the lumen with solutions with or without chloride, which was substituted by gluconate. This component was not different from zero in control and alkalotic (chronic plus acute) Wistar rats. Homozygous Brattleboro rats (BRB), genetically devoid of antidiuretic hormone, were used since this hormone has been shown to stimulate H⁺ secretion, which could mask bicarbonate secretion. In these rats, no evidence for Cl^–/HCO ₃ ^– exchange was found in control BRB and in early distal segments of alkalotic animals, but in late distal tubule a significant component of 0.14±0.033 nmol/cm² · sec was observed, which, however, is small when compared to the reabsorptive flow found in control Wistar rats, of 0.95±0.10 nmol/cm² · sec. In addition, 5×10^–4 m SITS had no effect on distal bicarbonate reabsorption in controls as well as on secretion in alkalotic Wistar and Brattleboro rats, which is compatible with the absence of effect of this drug on the apical Cl^–/HCO ₃ ^– exchange in other tissues. It is concluded that most distal alkalinization is not Cl^– dependent, and that Cl^–/HCO ₃ ^– exchange may be found in cortical distal tubule, but its magnitude is, even in alkalosis, markedly smaller than the reabsorptive flux, which predominates in the rats studied in this paper, keeping luminal pH lower than that of blood.     

The influence of amino acid starvation on the UV reversibility of the strains ofEscherichia coli B/rthy − trp − Hcr + andEscherichia coli B/rthy − trp − Hcr −

The fraction of inducedtrp ⁺ reversions in the strains ofEscherichia coli B/rthy ⁻ trp ⁻ Hcr ⁺ andEscherichia coli B/rthy ⁻ trp ⁻ Hcr ⁻ was studied in the course of starvation for an essential amino acid. UV light as a mutagenic factor was used. It was found that there is a decrease in the proportion of inducedtrp ⁺ reversions in the strain ofHcr ⁺ type during starvation. Such a decrease was however observed only with that fraction oftrp ⁺ reversions which is expressed in selective plates where several divisions of irradiated cells are caused. The proportion oftrp ⁺ reversions expressed on minimal plates does not change during starvation. With the strain ofHcr ⁻ type the proportion of inducedtrp ⁺ mutations remains unaltered irrespective of the nature of the selective plates.     

The accumulation of superoxide radical during the aerobic action of xanthine oxidase A requiem for H2O4−

The action of xanthine oxidase upon acetaldehyde or xanthine at pH 10.2 has been shown to be accompanied by substantial accumulation of O₂^? during the first few minutes of the reaction. H₂O₂ decreases this accumulation of O₂^? presumably because of the Haber-Weiss reaction ($\text{H}{}_2\text{O}{}_2\text{+}\text{O}{}_2{}^{\mathrm{?}}\text{→}\text{OH}{}^{\mathrm{?}}\text{+}\text{OH}\text{+}\text{O}{}_2$) and very small amounts of superoxide dismutase eliminate it. This accumulation of O₂^? was demonstrated in terms of a burst of reduction of cytochrome $\text{c}$, seen when the latter compound was added after aerobic preincubation of xanthine oxidase with its substrate. The kinetic peculiarities of the luminescence seen in the presence of luminol, which previously led to the proposal of H₂O₄^?, can now be satisfactorily explained entirely on the basis of known radical intermediates.     

Synthesis and evaluation of an 18F-labeled boramino acid analog of aminosuberic acid for PET imaging of the antiporter system xC−

In this study, we synthesized ¹⁸F-ASu-BF₃, a close boramino acid analog of 5-[¹⁸F]fluoro-aminosuberic acid (¹⁸F-ASu), via ¹⁸F-¹⁹F isotope exchange reaction and evaluated its potential for imaging with positron emission tomography (PET). ¹⁸F-ASu-BF₃ was stable in mouse plasma and taken up into PC3 prostate cancer cells via the system x_C^? amino acid transporter. The continuous use of isoflurane for anesthesia during dynamic imaging acquisition slowed down the excretion of ¹⁸F-ASu-BF₃ and enabled visualization of PC3 tumor xenografts in mice. In contrast, no tumor visualization was observed from static images of ¹⁸F-BF₃-ASu due to its rapid renal excretion mediated in part by the organic anion transporter. Our data indicate that the pharmacokinetics of amino acids could be altered after being converted into their boramino acid analogs. Therefore, care should be taken when using the boramino acid strategy to design and prepare ¹⁸F-labeled tracers for imaging amino acid transporters/receptors with PET.     

Effects of salicylate on HCO 3 − /Cl− exchange across the human erythrocyte membrane

Summary Changes in extracellular pH (pH _o ) in human red cell suspensions were monitored in a stopped-flow rapid reaction apparatus. A 20% suspension of washed human RBC in saline at pH 7 containing NaHCO₃ and extracellular carbonic anhydrase was mixed with an equal volume of buffered saline solution at pH 6.7. Sodium salicylate, when present, was added to both the erythrocyte suspension and the buffer solution. The effects of salicylate in the therapeutic to toxic concentration range on HCO ₃ ^– /Cl^– exchange were studied at 37°C. HCO ₃ ^– /Cl^– exchange flux was estimated using the extracellular buffer capacity and the difference betweendpH _o /dt using a control RBC suspension and that using a suspension of RBC whose anion exchange pathway was markedly inhibited. The results show that salicylate competitively decreases the rate of HCO ₃ ^– /Cl^– exchange, with inhibition increasing as salicylate concentration increases.K _I is 2.4mm. At a salicylate concentration of 10mm, HCO ₃ ^– /Cl^– exchange under the conditions of our experiments was inhibited by more than 70%. These findings are consistent with the possibility that CO₂ transfer in capillary bedsin vivo may be diminished in the presence of salicylate due to slowing of red cell HCO ₃ ^– /Cl^– exchange.     

Raman spectral analysis of the 1300 cm−1 region for lipid and membrane studies

The Raman spectra of fatty acids, fatty acid methyl esters and several membrane lipids are analyzed in the 1300 cm^?1 region. The ratio of peak intensities at 1303/1267 cm^?1 varies linearly with the ratio of methylene to vinyl groups in the hydrocarbon chain. This parameter should be useful for estimating the degree of unsaturation in isolated lipids and lipids in membranes.     

On the structure of the lipopolysaccharide core in the cell wall of Escherichia coli K12 W2252-11U− and its ter-mutant cells

From Escherichia coli K12 W2252-11U^? (wild type I and II) and its Ter-mutants, lipopolysaccharides were isolated and the primary structure of its core region was elucidated, the configuration of the glucose III (1→2)-linked to the glucose II residue in the core of parents (wild type I and II) changes into the configuration of the glucose (1→3)-linkage by the Ter-mutation. Also, the structure of the core in the Ter-15 mutant differs from that in the parent (wild type I) and is similar to that in Escherichia coli C used as an indicator for φ×174 phage. D-ribose residue in the core of the Ter-21 mutant links to the glucose I residue instead of the galactose residue in the core of the parent (wild type II).     

Redox reactions of the hydrogenase-cytochrome C3 system from Desulfovibrio gigas with the synthetic analogue of ferredoxin active sites [Fe4S4 (SCH2CH2OH)4]2−

The synthetic cluster [Fe₄S₄ (SCH₂CH₂OH)₄]^2? and ferredoxin I from $\text{Desulfovibrio}\text{gigas}$ have been comparatively reacted in the presence of hydrogen with two enzyme components isolated from the $\text{D.}\text{gigas}$ electron carrier system: hydrogenase and cytochrome c₃. The reactions have been followed by spectrophotometric and manometric methods. No reduction is detected unless both enzymes are simultaneously present. The easy reduction in the presence of hydrogenase-cytochrome c₃ combination takes place in two stages corresponding each to one equivalent of reduction. The first stage is reversible while the second is not and yields a super reduced species deriving from the cluster complex.     

Effect of concentration of iodide on the bound species of I2/I−3 in the amylose-iodine complex

A liquid-liquid distribution method, with heptane as the organic solvent, involving evaluation of the concentration of free 1⁻ by magnetic circular dichroism, has been developed for determining the bound amounts of I₂/I⁻₃ in the amylose-iodine complex in unbuffered aqueous solutions. The effect of I₂ and I⁻ concentrations on the bound species of iodine in the complex was investigated by using this method. We found that the stoichiometric bound species of I₂/I⁻₃ is independent of the concentration of I₂ at a given I⁻ concentration. However, the bound species strongly depends on I⁻ concentration, and varies from I⁻₃ at 10 mM KI to I⁻₁₅ at 0M KI. Moreover, the number of d-glucosyl residues required for including one iodine atom is within the range of 2.7 to 3.0, regardless of I⁻ concentration. It was concluded that the bound species are governed by the distribution of the actual species I₂·I₂ (I₄), (I₄), I₂·I⁻₃ (I⁻₅), and I⁻₃·I⁻₃ (I²⁻₆), which are responsible for the blue color of the complex.