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An Ussing chamber was used to demonstrate that synthetic amphiphilic anion transporters function as chloride transporters in mammalian airway epithelial cells.
1. A study has been made of the effects of ATP and alpha-oxoglutarate on the rate of metabolism of propionate by whole mitochondria from sheep liver, and by mitochondria disrupted with ultrasonic energy or by freezing and thawing. Whole mitochondria metabolized propionate aerobically; the rate was increased and stabilized by 0.5mm-ATP, and increased at least a further 50% by 1.67mm-alpha-oxoglutarate. 2. Anaerobically, externally added ATP at high concentrations permitted slow consumption of propionate. 3. In the presence of 1.3mm-ATP, but in the absence of alpha-oxoglutarate, there was no significant lag phase in the removal of propionate by whole mitochondria, and the rate declined at concentrations below 2mm. In the additional presence of 1.67mm-alpha-oxoglutarate or -glutamate, propionate was removed at linear rates until the residual propionate concentration was about 0.1mm. 4. Maximum rates of metabolism of propionate by whole mitochondria with 1.3mm-ATP occurred with alkali-metal chloride concentrations of 65-95mm and with K(+)/Na(+) ratios 5-10, both in the presence and absence of alpha-oxoglutarate. 5. With disrupted mitochondria stimulatory effects of alpha-oxoglutarate were obtained only aerobically, only with propionate and not propionyl-CoA as substrate, and only when sufficient mitochondrial structure remained to permit unsupplemented metabolism of propionate to occur. 6. In the presence of ATP and CoA, disrupted mitochondria fixed [2-(14)C]propionate at a rate adequate to explain the rate with whole mitochondria stimulated with ATP and alpha-oxoglutarate. 7. With both whole and partially disrupted mitochondria in the absence of ATP, the rate of metabolism of propionate was inhibited by about 80% by 3.3mm-AMP. The inhibition was partly overcome by alpha-oxoglutarate plus CoA. 8. It is concluded that the ultimate effect of alpha-oxoglutarate was to increase the rate of supply of ATP within the mitochondria. Reasons are given why it is premature to conclude that the extra ATP arose entirely from the oxidation of alpha-oxoglutarate itself.
CFTR is a cyclic AMP (cAMP)-activated chloride (Cl-) channel and a regulator of outwardly rectifying Cl- channels (ORCCs) in airway epithelia. CFTR regulates ORCCs by facilitating the release of ATP out of cells. Once released from cells, ATP stimulates ORCCs by means of a purinergic receptor. To define the domains of CFTR important for Cl- channel function and/or ORCC regulator function, mutant CFTRs with N- and C-terminal truncations and selected individual amino acid substitutions were created and studied by transfection into a line of human airway epithelial cells from a cystic fibrosis patient (IB3-1) or by injection of in vitro transcribed complementary RNAs (cRNAs) into Xenopus oocytes. Two-electrode voltage clamp recordings, 36Cl- efflux assays, and whole cell patch-clamp recordings were used to assay for the Cl- channel function of CFTR and for its ability to regulate ORCCs. The data showed that the first transmembrane domain (TMD-1) of CFTR, especially predicted alpha-helices 5 and 6, forms an essential part of the Cl- channel pore, whereas the first nucleotide-binding and regulatory domains (NBD1/R domain) are essential for its ability to regulate ORCCs. Finally, the data show that the ability of CFTR to function as a Cl- channel and a conductance regulator are not mutually exclusive; one function could be eliminated while the other was preserved.
The opening and closing of chloride (Cl-) channels in the ClC family are thought to tightly couple to ion permeation through the channel pore. In the prototype channel of the family, the ClC-0 channel from the Torpedo electric organ, the opening-closing of the pore in the millisecond time range known as "fast gating" is regulated by both external and internal Cl- ions. Although the external Cl- effect on the fast-gate opening has been extensively studied at a quantitative level, the internal Cl- regulation remains to be characterized. In this study, we examine the internal Cl- effects and the electrostatic controls of the fast-gating mechanism. While having little effect on the opening rate, raising [Cl-]i reduces the closing rate (or increases the open time) of the fast gate, with an apparent affinity of >1 M, a value very different from the one observed in the external Cl- regulation on the opening rate. Mutating charged residues in the pore also changes the fast-gating properties-the effects are more prominent on the closing rate than on the opening rate, a phenomenon similar to the effect of [Cl-]i on the fast gating. Thus, the alteration of fast-gate closing by charge mutations may come from a combination of two effects: a direct electrostatic interaction between the manipulated charge and the negatively charged glutamate gate and a repulsive force on the gate mediated by the permeant ion. Likewise, the regulations of internal Cl- on the fast gating may also be due to the competition of Cl- with the glutamate gate as well as the overall more negative potential brought to the pore by the binding of Cl-. In contrast, the opening rate of the fast gate is only minimally affected by manipulations of [Cl-]i and charges in the inner pore region. The very different nature of external and internal Cl- regulations on the fast gating thus may suggest that the opening and the closing of the fast gate are not microscopically reversible processes, but form a nonequilibrium cycle in the ClC-0 fast-gating mechanism.
Two groups of 10 healthy young men were matched on the basis of their free-choice consumptions of regular table salt. For 28 days they were then fed carefully controlled low-sodium foods. One group was permitted free-choice seasoning of these foods with regular table salt, the other with a 1:1 mixture of sodium and potassium chlorides. Intakes and urinary and fecal excretions of sodium and potassium were determined by analysis. The results were: 1) the subjects salted their food with essentially the same amounts of regular table salt as of the 1:1 mixture; 2) the sodium intake associated with seasoning at the table was reduced with the 1:1 mixture to 44 percent of the amount consumed with regular table salt; and 3) over a 28-day period there was no tendency for subjects to increase their use of a 1:1 mixture, when used in place of regular table salt, to compensate for their reduced sodium intakes. Under the conditions of this study, the use of the 1:1 mixture as a seasoning agent for foods that contained no added salt resulted in an average total intake of sodium (food plus seasoning) which was 55 percent that of the subjects using regular table salt.
Intracellular Cl- activity (aCli) and 5-hydroxytryptamine (5-HT)-induced membrane currents of Retzius neurons in the central nervous system of the medicinal leech were measured using Cl- sensitive microelectrodes and a two-microelectrode voltage-clamp technique. At the membrane of Retzius neurons Cl- ions were not passively distributed. Under different conditions the chloride equilibrium potential (ECl, -60.1 mV for isotonic saline and -57.8 mV for a hypertonic saline) was negative with respect to the membrane potential (Em, -55 +/- 3.8 and -47 +/- 3.4 mV respectively). The endogenous neurohormone 5-HT always polarized the membrane of Retzius neurons in the direction of ECl. When voltage-clamping the membrane of Retzius neurons near the resting potential both in situ and in primary culture, application of 5-HT produced an outward current (I5-HT) and an increase in membrane conductance. Current-voltage relationships for I5-HT showed a slight outward rectification and reversal potentials of -61.6 +/- 3.1 mV in situ and -66 +/- 3.1 mV in primary culture, both values being comparable to the ECl of Retzius neurons as measured in situ. The results indicate that 5-HT increases the Cl- conductance of Retzius neurons, thereby hyperpolarizing the cell membrane and affecting both the excitability of the neuron and 5-HT release from it. This could affect the feeding and swimming behaviour of the leech.
1. The relationship between cell volume and volume-sensitive Cl- conductance during hyposmotic cell swelling of patched cells and the effects of intracellular chloride on the conductance have been studied in mouse L-fibroblasts. To this end, swelling-activated current and cell volume were measured simultaneously in cells dialysed with low-Cl- (16 mM) or high-Cl- (130 mM) solutions using the whole-cell patch-clamp technique and videomicroscopy. 2. The increase in cell volume of patched cells and the volume-sensitive conductance saturated during a 4-5 min exposure to mildly hyposmotic solutions (15-20 % less than isosmotic). The swelling of patched cells varied considerably and was greater than the swelling of intact cells. No correlation between the maximal values of the volume-sensitive conductance and the maximal volumes of swollen cells was evident for cells dialysed with the low-Cl- solutions. 3. The amplitude of the volume-sensitive conductance decreased with a reduction in either extracellular or intracellular Cl- concentration; the size of the maximal conductance was not modulated by intracellular Cl- ions. 4. The activation of the volume-sensitive conductance was slower in high-Cl- cells than in low-Cl- cells whether it was induced by hypotonic cell swelling or by cell inflation; in low-Cl- cells the conductance saturated before the cell volume had reached its maximal value. 5. It is concluded that in patched cells an increase in cell volume triggers activation of the volume-sensitive Cl- conductance but does not determine its amplitude and that the rate of activation of the conductance is affected by the intracellular Cl- concentration.
CLC-3, a member of the CLC family of chloride channels, mediates function in many cell types in the body. The multifunctional calcium-calmodulin-dependent protein kinase II (CaMKII) has been shown to activate recombinant CLC-3 stably expressed in tsA cells, a human embryonic kidney cell line derivative, and natively expressed channel protein in a human colonic tumour cell line T84. We examined the CaMKII-dependent regulation of CLC-3 in a smooth muscle cell model as well as in the human colonic tumour cell line, HT29, using whole-cell voltage clamp. In CLC-3-expressing cells, we observed the activation of a Cl(-) conductance following intracellular introduction of the isolated autonomous CaMKII into the voltage-clamped cell via the patch pipette. The CaMKII-dependent Cl(-) conductance was not observed following exposure of the cells to 1 microm autocamtide inhibitory peptide (AIP), a selective inhibitor of CaMKII. Arterial smooth muscle cells express a robust CaMKII-activated Cl(-) conductance; however, CLC-3(-/-) cells did not. The N-terminus of CLC-3, which contains a CaMKII consensus sequence, was phosphorylated by CaMKII in vitro, and mutation of the serine at position 109 (S109A) abolished the CaMKII-dependent Cl(-) conductance, indicating that this residue is important in the gating of CLC-3 at the plasma membrane.
Several members of the CLC family of Cl- channels and transporters are expressed in vesicles of the endocytotic-lysosomal pathway, all of which are acidified by V-type proton pumps. These CLC proteins are thought to facilitate vesicular acidification by neutralizing the electric current of the H+-ATPase. Indeed, the disruption of ClC-5 impaired the acidification of endosomes, and the knock-out (KO) of ClC-3 that of endosomes and synaptic vesicles. KO mice are available for all vesicular CLCs (ClC-3 to ClC-7), and ClC-5 and ClC-7, as well as its beta-subunit Ostm1, are mutated in human disease. The associated mouse and human pathologies, ranging from impaired endocytosis and nephrolithiasis (ClC-5) to neurodegeneration (ClC-3), lysosomal storage disease (ClC-6, ClC-7/Ostm1) and osteopetrosis (ClC-7/Ostm1), were crucial in identifying the physiological roles of vesicular CLCs. Whereas the intracellular localization of ClC-6 and ClC-7/Ostm1 precluded biophysical studies, the partial expression of ClC-4 and -5 at the cell surface allowed the detection of strongly outwardly rectifying currents that depended on anions and pH. Surprisingly, ClC-4 and ClC-5 (and probably ClC-3) do not function as Cl- channels, but rather as electrogenic Cl--H+ exchangers. This hints at an important role for luminal chloride in the endosomal-lysosomal system.
Hereford x Angus crossbred steers (n = 36) were stunned, exsanguinated, and infused via the carotid artery either with an aqueous solution containing 98.52% water, 0.97% saccharides, 0.23% sodium chloride, and 0.28% phosphates (MPSC; n = 12) or with 0.3 M CaCl2 (n = 12). The remaining 12 steers served as noninfused controls. At 48 h postmortem, the quadriceps muscles and subcutaneous fat were removed from the carcasses, frozen, and later made into ground beef (18 to 20% fat). The longissimus lumborum (LL), semimembranosus, and psoas major (PM) also were removed, vacuum packaged, aged until 14 d postmortem, and then one steak was sliced from each muscle for visual and instrumental color evaluations. The inside (ISM) and outside (OSM) portions of the SM were evaluated separately. The LL and OSM steaks from MPSC-infused carcasses had a lighter red (P < 0.05) initial appearance than steaks from the other treatments. The LL steaks from noninfused carcasses had the most (P < 0.05) uniform color; the MPSC treatment was intermediate, and the CaCl2 treatment was the most two-toned. Steaks from both infusion treatments had higher (P < 0.05) L* values for the LL, ISM, and OSM muscles compared with noninfused carcasses. In general, the LL from CaCl2-infused carcasses had lower (P < 0.05) a* values, saturation indices, and 630 nm to 580 nm reflectance values, and had larger (P < 0.05) hue angles. Infusion with MPSC increased (P < 0.05) hue angles in the LL and OSM. Display color stability was lowest (P < 0.05) for LL steaks from CaCl2-infused carcasses, whereas steaks from MPSC-infused carcasses were lighter red in initial color, but otherwise had display color stability similar to those from noninfused carcasses. No differences (P > 0.05) due to infusion were found for any color traits for the PM muscle and ground beef. Carotid artery vascular infusion of carcasses with CaCl2 resulted in undesirable meat colors, whereas the MPSC solution lightened loin and inside round color in a desirable way, but the color stability was slightly less compared to muscle from noninfused carcasses. Infusion effects were not consistent among muscles, and further research will be needed to determine what caused these differences.
Mutant p53 protein and anti-p53 antibody in circulating blood can be detectedamong individuals with mutations of the p53 tumor suppressor gene. Plasma mutant p53 protein and anti-p53 antibody have also been associated with vinyl chloride monomer (VCM) exposure, although the mechanism of VCM-related carcinogenesis remains unclear. Polymorphisms of metabolic and DNA repair genes have been implicated in chemical exposure-related carcinogenesis. The aim of this study is to explore the association between polymorphisms of metabolic and DNA repair genes with mutant p53 protein and anti-p53 antibody expression induced by VCM. Study subjects comprised 333 male workers occupationally exposed to VCM. Plasma mutant p53 protein and anti-p53 antibody detected with ELISA were grouped together as p53 overexpression. Genotypes of cytochrome P450 2E1 (CYP2E1), aldehyde dehydrogenase 2 (ALDH2), glutathione S-transferase T1 (GSTT1), and X-ray repair cross-complementing group 1 (XRCC1, exon 10) genes were identified by the PCR. High VCM exposure group had significantly higher p53 overexpression as compared with low exposure group [odds ratio (OR), 2.1; 95% confidence interval (CI), 1.1-3.8]. Individuals having experienced a high VCM exposure and displaying a XRCC1 Gln-Gln genotype had a highest risk of p53 overexpression among those having different combinations of VCM exposure and XRCC1 genotypes (OR, 6.5; 95% CI, 1.7-24.2). Interestingly, those subjects reflecting a CYP2E1 c2c2 genotype among the low VCM-exposure group demonstrated a greater risk of p53 overexpression (OR, 9.8; 95% CI, 1.2-81.6) as compared with those experiencing a low VCM exposure and CYP2E1 c1c1/c1c2 genotypes. Additional analysis revealed that individuals possessing more susceptible XRCC1 Gln-Gln, CYP2E1 c2c2, ALDH2 1-2/2-2, and non-null GSTT1 genotypes were more likely to reveal p53 overexpression. Our results suggest that susceptible XRCC1 and CYP2E1 genotypes may modulate the mutation of the p53 gene among VCM-exposed workers.
Chloride ion (Cl-) effects on chloroperoxidase (CPO)-catalyzed peroxidation of catechol were used to probe the involvement of Cl- in CPO reactions. High concentrations of Cl- inhibit catechol peroxidation by competing with hydrogen peroxide (KI = 370 mM). However, at lower concentrations, Cl- is a linear competitive activator versus catechol (KDC = 35 mM). Addition of good halogenation substrates to the peroxidatic reaction mixture converts Cl- from a competitive activator to a competitive inhibitor. The KI (10 mM) for this halogenation substrate promoted Cl- inhibition is equivalent to the KM (11 mM) for Cl- in CPO-catalyzed halogenation reactions. During this inhibition, the halogenation substrate is consumed and, at the point where its consumption is complete, Cl- again becomes an activator. Also, at 2.0 mM hydrogen peroxide, CPOs chlorination reaction and its Cl- -activated peroxidatic reaction have similar apparent kcat values. All data are consistent with a mechanism in which Cl- competes with catechol for binding to CPO Compound I. Catechol binding initiates the Cl- -independent path, in which Compound I acts as the oxidizing agent for catechol. When Cl- binds to Compound I, it reacts to yield the enzymatic chlorinating intermediate which is responsible for either the oxidation of catechol in the Cl- -dependent path or the chlorination of substrates in the halogenation pathway. Cl- activation of the peroxidatic reaction is due to a shift from the Cl- -independent pathway to the Cl- -dependent process. The mechanism is unique in that exclusion of the substrate from its primary binding site leads to an increase in the catalytic efficiency of the reaction. This catechol-Cl- system also offers further potential for probing the specificity and chemistry of the key enzymatic intermediates in haloperoxidase-catalyzed reactions.
Poly Vinly Chloride (PVC) is a very durable, practical and economical plastic, which is so wide spread in its use that it may become difficult to replace it. The annual amount of world production has been constantly increasing and has reached the figure of 8.5m tons. While PVC is so useful, the health hazards which accompany this plastic are causing alarm in the industrially developed countries. The hazards are severe and include defects such as Haemangioendothelial sarcoma (Angiosarcoma) of the liver, Acro-Osteolysis of the fingers and Sclerodermic type skin lesions. This paper illustrates the recent developments, the clinical findings and the measures necessary to control the disease. Moreover, this paper should help alerting the health authorities in the developing countries about the health hazards involved in the manufacture and utilization of PVC. Due to the restrictions imposed on PVC producing plants, there is a real eventuality that these plants may be transferred to the developing countries, where this disease is still unknown.
The manufacture of PVC is necessary for supplying the demands of modern society, for maintaining the equilibrium in the chemical industry such as the manufacture of caustic soda solution-chlorine by means of rocksalt electrolysis, and for the safe disposal of chlorine for environmental reasons. The manufacture of vinyl chloride and dichloroethane is illustrated. The conventional processing of PVC into consumer goods, after treatment of rigid and plasticized PVC compounds or granulate, is demonstrated.
The heavy metal cadmium (Cd) causes hepatotoxicity upon acute administration. Kupffer cells, the resident macrophages of the liver, have been suggested to play a role in Cd-induced hepatotoxicity. Gadolinium chloride (GdCl3) may prevent Cd-induced hepatotoxicity by suppressing Kupffer cells. However, GdCl3 also induces the Cd-binding protein, metallothionein (MT). Therefore, this study was conducted to determine whether GdCl3 prevents Cd-induced hepatotoxicity via the induction of MT. Hepatic MT and Kupffer cell counts were analyzed 24 h after wild-type (WT) mice were administered saline or 10, 30, or 60 mg GdCl3/kg. GdCl3 induced MT in a dose-dependent manner without affecting nonprotein sulfhydryl content. All examined doses of GdCl3 were effective at eliminating Kupffer cells from the liver. To examine the hepatoprotective effects of GdCl3, WT and MT-null mice were pretreated with saline or 10, 30, or 60 mg GdCl3 24 h prior to a hepatotoxic dose of Cd (2.5 mg Cd/kg). Blood and livers were removed 16 h later and analyzed for hepatotoxicity as well as MT, Cd, and Kupffer cell content. Hepatotoxicity was alleviated in both WT and MT-null mice that were pretreated with 30 or 60 mg GdCl3/kg, indicating that MT induction is not required for the hepatoprotective effects of GdCl3. Hepatic Cd content was not decreased by GdCl3, demonstrating that GdCl3 does not negatively affect Cd distribution to the liver. Kupffer cells were depleted at all three doses of GdCl3, whereas hepatoprotection was only observed at doses of 30 and 60 mg GdCl3/kg. This does not rule out Kupffer cells in the mechanism of Cd-induced hepatotoxicity, but it does suggest that GdCl3 exerts hepatoprotective effects on the liver aside from depleting Kupffer cells. In summary, these data substantially rule out MT induction and decrease the importance of Kupffer cells as mechanisms of GdCl3-induced protection from Cd-induced hepatotoxicity.
Intracellular chloride activities, (Cl)c, in rabbit gallbladder were determined by using conventional (Kcl-filled) microelectrodes and Cl-selective, liquid ion-exchanger, microelectrodes. The results indicated that in the presence of a normal Ringer's solution, (Cl)c averages 35mM; this value is 2.3 times that predicted for an equilibrium distribution across the mucosal and baso-lateral membranes. On the other hand, when the tissue is bathed by Na-free solutions, (Cl)c declines to a value that does not differ significantly from that predicted for an equilibrium distribution. These results, together with those of Frizzell et al. (J. Gen. Physiol. 65:769, 1975) provide, for the first time, compelling evidence that (i) the movement of Cl from the mucosal solution into the cell is directed against an electrochemical potential difference (23mV); and (ii) this movement is energized by coupling to the entry of Na down a steep electrochemical potential difference. Finally, our data suggest that (i) Cl exit from the cell across the basolateral membrane may be coupled to the co-transport of a cation or the countertransport of an anion; and (ii) the mechanism responsible for active Na extrusion from the cell across the baso-lateral membrane is rheogenic (electrogenic), and is not the result of a neutral Na-K exchange.
Open-tip and Cl--selective microelectrodes were used to study the effect of external pH on apical membrane potential (Va) and intracellular chloride activity (aiCl) in epithelial cells of Necturus gallbladder. Increasing the pH from 7.2 to 8.2 in the mucosal, the serosal, or in both bathing solutions simultaneously, hyperpolarized Va (control value -60 +/- 5 mV) by about -6, -10 and -17 mV, respectively, but did not significantly change the transepithelial potential (VT = 0.3 +/- 0.5 mV). Identical hyperpolarizations were recorded with Cl--selective microelectrodes, even 40 min after changing external pH. Thus, aiCl (12 +/- 2 mM) remained essentially constant. The ratio fVa between the deflections in Va and VT produced by transepithelial current pulses, which is an approximate measure of the fractional resistance of the apical membrane, decreased when mucosal pH was increased, and increased when serosal pH was raised. The changes in Va and fVa are due, in part at least, to the known pH dependence of cell membrane K+ conductance (PK) in this tissue. The constancy of aiCl, despite significant increases in Va, indicates that cell membrane Cl- conductance (PCl) is virtually zero or decreases, with increased external pH, in a way that compensates for the increased driving force for Cl- exit. Experiments in which 90 mM gluconate or 90 mM methylsulfate were substituted for an equivalent amount of luminal Cl- did not provide any evidence for a significant contribution of Cl- ions, per se, to the emf or conductance of the apical membrane. They suggested, rather, a dependence of apical membrane cation permeability on luminal Cl- concentration. Since basolateral membrane PCl is known to be very low, the insensitivity of aiCl to Va is the consequence of a negligible electrodiffusive Cl- permeability at both cell membranes. Thus, overall, transcellular Cl- transport in Necturus gallbladder is, in large measure, effected by electroneutral processes.
Mercuric chloride (MC) and methylmercury (MeHg) were found to increase amino acid release from astrocytes. This suggests interaction with sulfhydryl (-SH) groups which are controlled by glutathione [GSH] levels. In the present study, we evaluated the effects of alterations in intracellular glutathione concentrations [GSH]i on the outcome of MC and MeHg treatment. [GSH]i were increased in a time-dependent fashion by incubating the astrocytes with 1 mM L-2-oxothiazolidine-4-carboxylic acid (OTC), a cysteine precursor. OTC attenuated the release of [2,3-3H]D-aspartic acid from astrocytes exposed to MC- (5 microM) and MeHg-(10 microM). MeHg-induced [3H]D-taurine release was also reduced by pretreatment of astrocytes with OTC. Treatment with BSO (50 microM) decreased [GSH]i in astrocytes, and increased [2,3-3H]D-aspartate release from MC- and MeHg-treated astrocytes, and [3H]D-taurine release from MeHg-treated cells. Neither OTC nor BSO when added to cultures in the absence of MC or MeHg had an effect on amino acid release by astrocytes. The current study underscores both the sensitivity of astrocytes to mercurials in terms of amino acid release and the relationship of these effects of astrocytic [GSH]i.