Characterization of a Na+-dependent betaine transporter with Cl- channel properties in squid motor neurons.
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Most marine invertebrates, including squids, use transporters to accumulate organic osmolytes such as betaine, to prevent water loss when exposed to elevated salinity. Although a limited number of flux studies have shown the Na+ dependence of betaine transport, nothing is known about the electrogenic properties of osmolyte transporters. We used whole cell and perforated-patch voltage-clamp techniques to characterize the electrical properties of the betaine transporter in giant fiber lobe motor neurons of the squid Lolliguncula brevis. Betaine activated a large, Cl--selective current that was reversibly blocked by 100 microM niflumic acid (97 +/- 2% block after 40 s, SD; n = 7) and partially inhibited by 500 microM SITS (29 +/- 11%; n = 5). The Cl- current was Na+ dependent and was virtually eliminated by isotonic replacement of Na+ with Li+, NMDG+, or Tris+. Concentration-response data revealed an EC50 in a physiologically relevant range for these animals of 5.1 +/- 0.9 mM (n = 11). In vertebrates, the betaine transporter is structurally related to the GABA transporter, and although GABA did not directly activate the betaine-induced current, it reversibly reduced betaine responses by 34 +/- 14% (n = 8). Short-term changes in osmolality alone did not activate the Cl- current, but when combined with betaine, Cl- currents increased in hypertonic solutions and decreased in hypotonic solutions. Activation of the betaine transporter and Cl- current in hypertonic conditions may affect both volume regulation and excitability in L. brevis motor neurons. This study is the first report of a novel betaine transporter in neurons that can act as a Cl- channel. (+info)
Na+-K+-2Cl- cotransporter in immature cortical neurons: A role in intracellular Cl- regulation.
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Na+-K+-2Cl- cotransporter has been suggested to contribute to active intracellular Cl- accumulation in neurons at both early developmental and adult stages. In this report, we extensively characterized the Na+-K+-2Cl- cotransporter in primary culture of cortical neurons that were dissected from cerebral cortex of rat fetus at embryonic day 17. The Na+-K+-2Cl- cotransporter was expressed abundantly in soma and dendritic processes of cortical neurons evaluated by immunocytochemical staining. Western blot analysis revealed that an approximately 145-kDa cotransporter protein was present in cerebral cortex at the early postnatal (P0-P9) and adult stages. There was a time-dependent upregulation of the cotransporter activity in cortical neurons during the early postnatal development. A substantial level of bumetanide-sensitive K+ influx was detected in neurons cultured for 4-8 days in vitro (DIV 4-8). The cotransporter activity was increased significantly at DIV 12 and maintained at a steady level throughout DIV 12-14. Bumetanide-sensitive K+ influx was abolished completely in the absence of either extracellular Na+ or Cl-. Opening of gamma-aminobutyric acid (GABA)-activated Cl- channel or depletion of intracellular Cl- significantly stimulated the cotransporter activity. Moreover, the cotransporter activity was elevated significantly by activation of N-methyl-D-aspartate ionotropic glutamate receptor via a Ca2+-dependent mechanism. These results imply that the inwardly directed Na+-K+-2Cl- cotransporter is important in active accumulation of intracellular Cl- and may be responsible for GABA-mediated excitatory effect in immature cortical neurons. (+info)
Cl- channels in basolateral TAL membranes. XIV. Kinetic properties of a basolateral MTAL Cl- channel.
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BACKGROUND: This article reports studies on the kinetics of chloride (Cl-) conductance in Cl- channels fused into bilayers from basolaterally enriched vesicles from rabbit outer medulla. A considerable body of evidence indicates that these channels represent rbClC-Ka, a 77 kDa kidney-specific protein of the ClC family of Cl- channels. rbClC-Ka, a candidate channel for mediating net Cl- absorption in the medullary thick ascending limb (MTAL), has been cloned from rabbit outer medulla and localized by immunofluorescence to basolateral membranes of the MTAL. Thus, this is the first account, to our knowledge, of the kinetics of ion permeation through a renal Cl- channel mediating net basolateral Cl- absorption in the thick ascending limb of Henle (TALH), and this channel may represent rbClC-Ka. METHODS: The electrophysiological properties of these channels were studied by fusing basolaterally enriched MTAL vesicles into planar bilayer membranes. RESULTS: Cl- conductance through these channels was concentration dependent and saturable. The relationship between gCl (pS) and symmetrical aqueous Cl- concentrations could be expressed in terms of the Michaelis equation with a limiting conductance (GClmax, pS) of 114 pS at infinitely high aqueous Cl- concentrations and a K1/2 of 163 mM Cl-. A log-log plot of the conductance-Cl- concentration relations, in the nonsaturating Cl- concentration range, had a slope of 0.91, that is, virtually unity. The relatively impermeant anion I- produced a voltage-dependent conductance blockade that could be overcome at high electric field strengths. CONCLUSIONS: The experimental data described earlier here fulfill the traditional criteria for a first-order process with a single Cl- ion occupying these channels at a given time. Although the channels may contain multiple ion binding sites, the latter function, in integral kinetic terms, as a single rate-limiting locus. (+info)
Inhibition of GABA-gated chloride channels by 12,14-dichlorodehydroabietic acid in mammalian brain.
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1. 12,14-dichlorodehydroabietic acid (12,14-Cl2DHA) reduced GABA-stimulated uptake of 36Cl- into mouse brain synaptoneurosomes suggesting inhibition of mammalian GABA(A) receptor function. 2. 12,14-Cl2DHA did not affect the binding of [3H]-muscimol to brain membranes but displaced specifically bound [3H]-EBOB. The inhibitory effect on [3H]-EBOB binding was not reversible. 12,14-Cl2DHA reduced the availability of [3H]-EBOB binding sites (Bmax) without changing the KD of the radioligand for remaining sites. 12,14-Cl2DHA did not affect the rate of association of [3H]-EBOB with its chloride channel receptor, but increased the initial rate of [3H]-EBOB dissociation. 3. 12,14-Cl2DHA enhanced the incidence of EPSCs when rapidly applied to cultured rat cortical neurones. Longer exposures produced block of IPSCs with marked increases in the frequency of EPSCs and min EPSCs. 12,14-Cl2DHA also irreversibly suppressed chloride currents evoked by pulses of exogenous GABA in these cells. 4. Ultimately, 12,14-Cl2DHA inhibited all synaptic traffic and action currents in current clamped cells indicating that, in contrast to picrotoxinin (which causes paroxysmal bursting), it is not fully selective for the GABA(A) receptor-chloride channel complex. 5. The depolarizing block seen with 12,14-Cl2DHA in amphotericin-perforated preparations implicates loss of Ca2+ buffering in the polarity change and this may account for inhibition of spontaneous action potentials. 6. Our investigation demonstrates that 12,14-Cl2DHA blocks GABA-dependent chloride entry in mammalian brain and operates as a non-competitive insurmountable GABA(A) antagonist. The mechanism likely involves either irreversible binding of 12,14-Cl2DHA to the trioxabicyclooctane recognition site or a site that is allosterically coupled to it. We cannot exclude, however, the possibility that 12,14-Cl2DHA causes localized proteolysis or more extensive conformational change within a critical subunit of the chloride channel. (+info)
The haemin storage (Hms+) phenotype of Yersinia pestis is not essential for the pathogenesis of bubonic plague in mammals.
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The haemin storage (Hms+) phenotype of Yersinia pestis enables this bacillus to form greenish/brown or red colonies on haemin or Congo Red agar plates, respectively, at 26 but not 37 degrees C. Escherichia coli strains that contain mutations in genes essential for siderophore biosynthesis, porphyrin generation and/or haemin transport remain unable to utilize exogenous haemin as a nutritional iron or porphyrin source when transformed with the cloned Y. pestis hmsHFRS locus. Further physiological analysis of the Hms+ phenotype of Y. pestis strain KIM6+ suggests that the haemin and inorganic iron stored by the Hms system was not used nutritionally under subsequent iron-deficient conditions. In vitro analysis of the bactericidal effects of hydrogen peroxide, superoxide and nitric oxide showed that Hms- Y. pestis cells, in certain cases, were more susceptible than the Hms+ parent cells to these reactive oxygen species at 26 and/or 37 degrees C. In adherence assays, a higher percentage of Hms+ cells were associated with HeLa cells and normal human neutrophils, compared to Hms- cells. However, the Hms+ phenotype did not provide any additional protection against the killing effects of neutrophils. Finally, LD50 analysis in subcutaneously infected mice showed that an Hms- strain was slightly more virulent than Hms+, indicating that the Hms phenotype is not essential for the pathogenesis of bubonic plague in mammals. (+info)
Effects of carbon dioxide inhalation on hematology, coagulation, and serum clinical chemistry values in rats.
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Blood samples from adult male and female Charles River Crl:CD (SD) BR rats were collected at weekly intervals for 4 wk to evaluate the effects of inhalation of an anesthetic dose of carbon dioxide (CO2) or of a carbon dioxide-oxygen mixture (CO2/O2) on hematology, coagulation, and serum biochemistry values. During the first 3 wk of the study, rats were assigned to 1 of 3 groups and were bled from the orbital sinus once weekly. Prior to the blood collection, rats in group 1 were exposed to room air only, rats in group 2 received CO2/O2 (approximately 66%:34% CO2:O2) by inhalation, and rats in group 3 received 100% CO2 by inhalation. In the rats exposed to CO2/O2 or CO2, leukocyte counts, lymphocyte counts, and glucose values were higher, and aspartate aminotransferase, creatine kinase, and calcium values were lower compared with those of rats exposed to room air only. Rats exposed to 100% CO2 had slightly (but statistically significant) lower mean corpuscular hemoglobin concentration when compared with rats exposed only to room air. During week 4, all rats were reassigned to 1 of 2 groups and were bled terminally via closed cardiac puncture following exposure to either CO2/O2 or CO2. Increased lymphocyte counts (males only) and glucose and chloride concentrations were noted for rats exposed to CO2/O2 compared with those exposed to CO2. These alterations reiterate the importance of comparing clinical pathology values to those of concurrent control groups that have experienced blood collection under identical conditions in order to avoid potential errors in the interpretation of data. (+info)
Cholinergic modulation of neostriatal output: a functional antagonism between different types of muscarinic receptors.
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It is demonstrated that acetylcholine released from cholinergic interneurons modulates the excitability of neostriatal projection neurons. Physostigmine and neostigmine increase input resistance (RN) and enhance evoked discharge of spiny projection neurons in a manner similar to muscarine. Muscarinic RN increase occurs in the whole subthreshold voltage range (-100 to -45 mV), remains in the presence of TTX and Cd2+, and can be blocked by the relatively selective M1,4 muscarinic receptor antagonist pirenzepine but not by M2 or M3 selective antagonists. Cs+ occludes muscarinic effects at potentials more negative than -80 mV. A Na+ reduction in the bath occludes muscarinic effects at potentials more positive than -70 mV. Thus, muscarinic effects involve different ionic conductances: inward rectifying and cationic. The relatively selective M2 receptor antagonist AF-DX 116 does not block muscarinic effects on the projection neuron but, surprisingly, has the ability to mimic agonistic actions increasing RN and firing. Both effects are blocked by pirenzepine. HPLC measurements of acetylcholine demonstrate that AF-DX 116 but not pirenzepine greatly increases endogenous acetylcholine release in brain slices. Therefore, the effects of the M2 antagonist on the projection neurons were attributable to autoreceptor block on cholinergic interneurons. These experiments show distinct opposite functions of muscarinic M1- and M2-type receptors in neostriatal output, i.e., the firing of projection neurons. The results suggest that the use of more selective antimuscarinics may be more profitable for the treatment of motor deficits. (+info)
Secondary radicals derived from chloramines of apolipoprotein B-100 contribute to HOCl-induced lipid peroxidation of low-density lipoproteins.
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Oxidation of low-density lipoproteins (LDL) is thought to contribute to atherogenesis. Although there is increasing evidence for a role of myeloperoxidase-derived oxidants such as hypochlorite (HOCl), the mechanism by which HOCl modifies LDL remains controversial. Some studies report the protein component to be the major site of attack, whereas others describe extensive lipid peroxidation. The present study addresses this controversy. The results obtained are consistent with the hypothesis that radical-induced oxidation of LDL's lipids by HOCl is a secondary reaction, with most HOCl consumed via rapid, non-radical reaction with apolipoprotein B-100. Subsequent incubation of HOCl-treated LDL gives rise to lipid peroxidation and antioxidant consumption in a time-dependent manner. Similarly, with myeloperoxidase/H2O2/Cl- (the source of HOCl in vivo), protein oxidation is rapid and followed by an extended period of lipid peroxidation during which further protein oxidation does not occur. The secondary lipid peroxidation process involves EPR-detectable radicals, is attenuated by a radical trap or treatment of HOCl-oxidized LDL with methionine, and occurs less rapidly when the lipoprotein was depleted of alpha-tocopherol. The initial reaction of low concentrations of HOCl (400-fold or 800-fold molar excess) with LDL therefore seems to occur primarily by two-electron reactions with side-chain sites on apolipoprotein B-100. Some of the initial reaction products, identified as lysine-residue-derived chloramines, subsequently undergo homolytic (one-electron) reactions to give radicals that initiate antioxidant consumption and lipid oxidation via tocopherol-mediated peroxidation. The identification of these chloramines, and the radicals derived from them, as initiating agents in LDL lipid peroxidation offers potential new targets for antioxidative therapy in atherogenesis. (+info)