Cl Uptake Research Articles

Ion uptake in Pinus banksiana treated with sodium chloride and sodium sulphate.

Within its wide range across Canada, jack pine is exposed to salinity from both natural and anthropogenic sources. To compare the effects of Cl and SO(4) on salt injury, sand and solution-culture grown jack pine (Pinus banksiana Lamb.) seedlings were treated with nutrient solutions containing 60 or 120 mM NaCl, 60 mM Na(2)SO(4), or a mixture of 60 mM NaCl and 30 mM Na(2)SO(4). After 5 weeks of salt treatments, concentrations of Cl, K, Na, and SO(4) were determined in roots, stem and needles of the current and previous years growth, and in necrotic needles. To determine the role of water uptake in the absorption and translocation of salts in plants, total transpiration was measured as the loss of water from a sealed system and related to total plant uptake of Cl, Na, and SO(4). Sodium uptake and root-to-shoot transport rates were greater in treatments containing Cl. A delay in root-to-shoot transport of both Na and Cl indicates retention of these ions in the roots. Electrolyte leakage of needles was more closely related to treatment Cl concentrations than treatment Na concentrations. The transport of Na ions to the shoot was related to the presence of Cl, but was not related to transpiration rate.

Influence of oxygenated fuel additives and their metabolites on γ-aminobutyric acid A (GABA A) receptor function in rat brain synaptoneurosomes

Experimental and occupational inhalational exposure to oxygenate fuel additives in reformulated gasoline has been reported to induce neurological symptoms (e.g., headache, nausea, dizziness). We reported previously that the ether additives (methyl- t-butyl ether (MTBE), t-amyl-methyl ether (TAME) and ethyl- t-butyl ether (ETBE)) and their metabolites ( t-amyl alcohol (TAA), t-butyl alcohol (TBA) and ethanol) alter the binding of [ 3H] t-butylbicycloorthobenzoate ([ 3H]TBOB), a ligand for the γ-aminobutyric acid A (GABA A) receptor in rat brain membrane preparations. To more directly assess the effects of the ethers and their alcohol precursors on GABA A receptor function, the uptake of 36Cl − was measured in synaptoneurosomes, a preparation of closed membrane sacs comprised of pre- and postsynaptic membranes from adult rat cerebral cortex. Each of the compounds caused a concentration-dependent enhancement of muscimol-stimulated uptake of 36Cl −, which diminished with further increasing concentrations. The potency of the enhancement by the compounds was in the rank order: MTBE=TAME>TAA=ETBE>TBA>ethanol. The half-maximally effective concentration (EC 50) for the facilitation of muscimol-stimulated 36Cl − uptake ranged from 0.06 to 3 mM, and that for the higher-dose inhibitory effect (IC 50) ranged from 3 to 50 mM. The facilitatory concentrations of the compounds are in the range of the blood concentrations reported in experimental animals after exposures known to induce CNS effects such as ataxia. The results suggest a potential role of the GABA A receptor in some of the reported neurotoxic effects of gasoline additives.

Unique regulation of anion/HCO3- exchangers by constitutive nitric oxide in rabbit small intestine

In the rabbit small intestine, there are three functionally different brush-border membrane (BBM) anion/HCO3- exchangers: 1) Cl/HCO3- exchange on the BBM of villus cells responsible for coupled NaCl absorption; 2) Cl/HCO3- exchange on the BBM of crypt cells possibly involved in HCO3- secretion; and 3) short-chain fatty acid (SCFA)/HCO3- exchange on the BBM of villus cells, which facilitates SCFA absorption. Although constitutive nitric oxide (cNO) has been postulated to alter many gastrointestinal tract functions, how cNO may specifically alter these three transporters is unknown. Inhibition of cNO synthase with NG-nitro-L-arginine methyl ester (L-NAME) 1) did not affect villus cell BBM Cl/HCO3 change, 2) stimulated crypt cell BBM Cl/HCO3- exchange, and 3) inhibited villus cell BBM SCFA/HCO3- exchange. D-NAME, an inactive analog of L-NAME, and L-N6-(1-iminoethyl)lysine, a more selective inhibitor of inducible NO, did not affect these transport processes. Kinetic studies demonstrated that 1) the mechanism of inhibition of crypt cell BBM Cl/HCO3- exchange is secondary to a decrease in the maximal rate of uptake of Cl, without an alteration in the affinity of the transporter for Cl, and 2) the mechanism of stimulation of villus cell BBM SCFA/HCO3- exchange is secondary to an increase in the affinity of the transporter for SCFA without an alteration in the maximal rate of uptake of SCFA. These results indicate that cNO uniquely regulates the three BBM anion/HCO3- transporters in the rabbit small intestine.

Net uptake of chloride across the posterior gills of the Chinese crab ( Eriocheir sinensis)

Two methods are commonly used for the determination of transbranchial net fluxes of Na + and Cl −: direct analysis of changes in ion concentrations in the external medium using flame spectrophotometry or titration (net flux method), and measurement of unidirectional ion fluxes by means of radioactive tracers (tracer method). When we applied both methods in the same preparation, the isolated perfused posterior gill of freshwater-acclimated Eriocheir sinensis, to determine net fluxes of Cl −, the results differed substantially. In artificial fresh water (AFW) containing NaCl, the net flux method yielded a net uptake, but the tracer method showed a net efflux of Cl −. The net uptake of Cl − was abolished in Na +-free AFW indicating that Cl − uptake is coupled with the uptake of Na +. Applying the tracer method, net efflux of Cl − remained almost unchanged in Na +-free AFW. This suggests the opposite mechanism, i.e. uncoupled uptake of Na + and Cl −. The discrepancy in the results obviously depends on the method employed. Since the data obtained with the net flux method explain the osmoregulatory performance of crabs living in fresh water, we consider this method as appropriate for determining net transbranchial ion fluxes.

Cl(-)/HCO(3)(-) exchange is acetazolamide sensitive and activated by a muscarinic receptor-induced [Ca(2+)](i) increase in salivary acinar cells.

Large volumes of saliva are generated by transepithelial Cl(-) movement during parasympathetic muscarinic receptor stimulation. To gain further insight into a major Cl(-) uptake mechanism involved in this process, we have characterized the anion exchanger (AE) activity in mouse serous parotid and mucous sublingual salivary gland acinar cells. The AE activity in acinar cells was Na(+) independent, electroneutral, and sensitive to the anion exchange inhibitor DIDS, properties consistent with the AE members of the SLC4A gene family. Localization studies using a specific antibody to the ubiquitously expressed AE2 isoform labeled acini in both parotid and sublingual glands. Western blot analysis detected an approximately 170-kDa protein that was more highly expressed in the plasma membranes of sublingual than in parotid glands. Correspondingly, the DIDS-sensitive Cl(-)/HCO(3)(-) exchanger activity was significantly greater in sublingual acinar cells. The carbonic anhydrase antagonist acetazolamide markedly inhibited, whereas muscarinic receptor stimulation enhanced, the Cl(-)/HCO(3)(-) exchanger activity in acinar cells from both glands. Intracellular Ca(2+) chelation prevented muscarinic receptor-induced upregulation of the AE, whereas raising the intracellular Ca(2+) concentration with the Ca(2+)-ATPase inhibitor thapsigargin mimicked the effects of muscarinic receptor stimulation. In summary, carbonic anhydrase activity was essential for regulating Cl(-)/HCO(3)(-) exchange in salivary gland acinar cells. Moreover, muscarinic receptor stimulation enhanced AE activity through a Ca(2+)-dependent mechanism. Such forms of regulation may play important roles in modulating fluid and electrolyte secretion by salivary gland acinar cells.

Rotavirus infection stimulates the Cl- reabsorption process across the intestinal brush-border membrane of young rabbits.

Rotavirus is a major cause of infantile gastroenteritis worldwide. However, the mechanisms underlying fluid and electrolyte secretion associated with diarrhea remain largely unknown. We investigated the hypothesis that loss of Cl(-) into the luminal contents during rotavirus infection may be caused by a dysfunction in the chloride absorptive capacity across the intestinal brush-border membrane (BBM). The luminal Cl(-) concentrations in the entire small intestine of young rabbits infected with lapine rotavirus decreased at 1 and 2 days postinfection (dpi), indicating net Cl(-) absorption. At 7 dpi, luminal Cl(-) concentrations were slightly increased, indicating a moderate net Cl(-) secretion. By using a rapid filtration technique, (36)Cl uptake across BBM was quantified by modulating the alkali-metal ion, electrical, chloride, and/or proton gradients. Rotavirus infection caused an identical, 127% +/- 24% increase in all Cl(-) uptake activities (Cl(-)/H(+) symport, Cl(-) conductance, and Cl(-)/anion exchange) observed across the intestinal BBM. The rotavirus activating effects on the symporter started at 1 dpi and persisted up to 7 dpi. Kinetic analyses revealed that rotavirus selectively affected the capacity parameter characterizing the symporter. We report the novel observation that rotavirus infection stimulated the Cl(-) reabsorption process across the intestinal BBM. We propose that the massive Cl(-) reabsorption in villi could partly overwhelm chloride secretion in crypt cells, which possibly increases during rotavirus diarrhea, the resulting imbalance leading to a moderate net chloride secretion.

Characterization of Cl-HCO3 exchange in basolateral membrane of rat distal colon.

Sodium-independent Cl movement (i.e., Cl-anion exchange) has not previously been identified in the basolateral membranes of rat colonic epithelial cells. The present study demonstrates Cl-HCO3 exchange as the mechanism for 36Cl uptake in basolateral membrane vesicles (BLMV) prepared in the presence of a protease inhibitor cocktail from rat distal colon. Studies of 36Cl uptake performed with BLMV prepared with different types of protease inhibitors indicate that preventing the cleavage of the COOH-terminal end of AE2 protein by serine-type proteases was responsible for the demonstration of Cl-HCO3 exchange. In the absence of voltage clamping, both outward OH gradient (pHout/pHin: 7.5/5.5) and outward HCO3 gradient stimulated transient 36Cl uptake accumulation. However, voltage clamping with K-ionophore, valinomycin, almost completely (87%) inhibited the OH gradient-driven 36Cl uptake, whereas HCO3 gradient-driven 36Cl uptake was only partially inhibited (38%). Both electroneutral HCO3 and OH gradient-driven 36Cl uptake were 1) completely inhibited by DIDS, an anion exchange inhibitor, with a half-maximal inhibitory constant (Ki) of approximately 26.9 and 30.6 microM, respectively, 2) not inhibited by 5-nitro-2-(3-phenylpropylamino)benzoic acid(NPPB), a Cl channel blocker, 3) saturated by increasing extravesicular Cl concentration with a Km for Cl of approximately 12.6 and 14.2 mM, respectively, and 4) present in both surface and crypt cells. Intracellular pH (pHi) was also determined with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-acetomethylester (BCECF-AM) in an isolated superfused crypt preparation. Removal of Cl resulted in a DIDS-inhibitable increase in pHi both in HCO3-buffered and in the nominally HCO3-free buffered solutions (0.28 +/- 0.02 and 0.11 +/- 0.02 pH units, respectively). We conclude that a carrier-mediated electroneutral Cl-HCO3 exchange is present in basolateral membranes and that, in the absence of HCO3, Cl-HCO3 exchange can function as a Cl-OH exchange and regulate pHi across basolateral membranes of rat distal colon.

Rat down-regulated-in-adenoma (rDRA) cDNA expresses a DIDS-insensitive and pH dependent Cl uptake in HEK293 cells

Rat down-regulated-in-adenoma (rDRA) cDNA expresses a DIDS-insensitive and pH dependent Cl uptake in HEK293 cells

Tolerance of citrus rootstock seedlings to saline stress based on their ability to regulate ion uptake and transport.

Forty-five-day-old seedlings of sour orange (Citrus aurantium L.) and Citrus macrophylla Wester, the most commonly used rootstocks in lemon orchards, were grown in nutrient solutions containing 1 (control), 10, 20, 30 or 60 mM NaCl for 14 days. The effects of salinity on growth, uptake, transport and accumulation of Cl- and Na+ ions in leaves, stem and four root segments were studied. The 60 mM NaCl treatment reduced leaf dry mass more in C. macrophylla (40%) than in sour orange (20%), whereas it reduced root dry mass more in sour orange (36%) than in C. macrophylla (20%). In C. macrophylla, Cl- and Na+ uptake rates were high at the beginning of the saline treatments, but low at the end of the 14-day experiment. In contrast, sour orange showed high uptake rates at the beginning and end of the experiment. In response to increasing salinity, root and shoot concentrations of Cl- and Na+ increased in sour orange, but not in C. macrophylla. Different loading characteristics of Cl- and Na+ were observed between young and old segments of the root system. In general, old root segments reached quasi-steady-states later than young root segments. These results suggest that sour orange and C. macrophylla have different regulatory mechanisms for uptake and transport of Cl- and Na+.

Salt tolerance of mango rootstocks (Magnifera indica L. cv. Osteen)

The aim of this work was to establish the criteria to determine the relative tolerance of mango rootstocks (Mangiferaindica L.) to high Cl- and Na+ concentrations present in irrigation water. Two rootstocks, Gomera-1 and Gomera-3 grafted with the cultivar Osteen, were subjected to study. Grafted plants were exposed to salinized irrigation watersmeasured by electrical conductivity (1.02, 1.50, 2.00 and 2.50 dS m-1). Results indicated differences in retainingtoxic elements in different organs (roots, stem, or leaves) of both rootstocks. Gomera-3 was more sensitive as took uphigher amounts of Cl- and Na+ than Gomera-1. Gomera-1 was more tolerant, being the tolerance possibly associatedwith the capacity of this rootstock to restrict the uptake and transport of Cl- and Na+ ions from the root system to theaboveground parts. The Cl- ions were more toxic in rootstock and cultivar leaves, while the Na+ ions were more toxicin the roots. Therefore, the present study reveals that Gomera-1 proved to be the most adaptable rootstock to salineconditions, making it feasible for use in areas with low water quality.

Decisive role of electronic polarization of the protein environment in determining the absorption maximum of halorhodopsin.

It is known that the absorption maximum of halorhodopsin is red shifted by 10 nm with the uptake of a chloride ion Cl(-). According to the X-ray structure, the ion is located at the position of the counterion of the chromophore, protonated retinal Schiff base. Thus, the direction of the observed spectral change is opposite to that expected from the pi-electron redistribution (an increase in the bond alternation) induced by the counterion. The physical origin of this abnormal shift is never explained in terms of any simple chemical analogues. We successfully explain this phenomenon by a QM/MM type of excitation energy calculation. The three-dimensional structure of the protein is explicitly taken into account using the X-ray structure. We reveal that the electronic polarization of the protein environment plays an essential role in tuning the absorption maximum of halorhodopsin.

Deletion of GABAA receptor alpha 1 subunit-containing receptors alters responses to ethanol and other anesthetics.

GABA(A) receptors have been implicated in mediating several acute effects of ethanol including anxiolysis, ataxia, sedation/hypnosis, and anticonvulsant activity. Ethanol sensitivity of neurons has been associated with expression of alpha1 subunit-containing receptors. The objective of this study was to determine the contribution of alpha1 subunit containing receptors to ethanol responses in comparison to neurosteroids and other anesthetics using GABA(A) receptor alpha1 subunit knockout mice. Deletion of alpha1 subunit-containing receptors did not alter the anxiolytic, ataxic, anticonvulsant, or hypnotic effects of ethanol or acute functional tolerance to ethanol but did increase sensitivity to the locomotor-stimulating effects of ethanol. The ability of ethanol to potentiate muscimol-stimulated chloride uptake and ethanol clearance was also not altered following alpha1 subunit deletion. The anticonvulsant and hypnotic effects of neurosteroids as well as their potentiating effect on GABA-mediated Cl(-) uptake were unaltered in alpha1(-/-) mice. The hypnotic effect of pentobarbital, etomidate, and midazolam were reduced, whereas the effect of ketamine was enhanced in alpha1(-/-) mice. Thus, GABA(A) receptor alpha1 subunit-containing receptors appear to influence the motor-stimulating effect of ethanol and the sedative/hypnotic effects of some anesthetics, but not ethanol. These receptors do not appear to be necessary for most ethanol responses, suggesting involvement of other GABA(A) receptor subtypes or other targets altogether.

Dilute culture media as an environmental or physiological simulant in cultured gill epithelia from freshwater rainbow trout.

The electrophysiological and ion-transporting properties of cultured gill epithelia from freshwater (FW) rainbow trout were examined in the presence of dilute cell culture media as an environmental or physiological simulant. Gill epithelia were cultured on cell culture inserts under symmetrical conditions (L15 apical-L15 basolateral) for 6-7 d. The following experiments were then conducted. (1) To mimic a gradual lowering of environmental salinity, apical L15 medium was progressively diluted with FW (first to 2/3 L15 for 8 h and then to 1/3 L15 for 6 h) before the introduction of apical FW (FW apical-L15 basolateral, analogous to a fish in a natural FW environment). Dilute apical media had no significant effect on the electrophysiological properties of preparations compared with symmetrical culture conditions, and no evidence for active Na(+) or Cl(-) transport was observed. Preparations subsequently exposed to apical FW exhibited a negative transepithelial potential and evidence of active Cl(-) uptake and slight Na(+) extrusion. (2) To mimic the extracellular fluid dilution that occurs in euryhaline fish after abrupt transfer from saline to FW, the osmolality or ionic strength (or both) of basolateral media was reduced by 20-40% (using either FW or FW + mannitol) while simultaneously replacing apical media with FW. Under these conditions, Na(+) and Cl(-) influx rates were low compared with efflux rates, while the Ussing flux ratio analysis generally indicated active Cl(-) uptake and Na(+) extrusion. The Na(+)-K(+) adenosine triphosphatase activity was not affected by alterations in basolateral osmolality. Our studies indicate that cultured trout gill epithelia are tolerant of media dilution from both the apical and the basolateral direction; however, neither treatment alone appeared to increase ion influx rates or stimulate active Na(+) uptake in cultured trout gill epithelia.

Influence of saline irrigation on growth, ion accumulation and partitioning, and leaf gas exchange of carrot (Daucus carota L.).

Like those of many horticultural crop species, the growth and leaf gas exchange responses of carrot (Daucus carota L.) to salinity are poorly understood. In this study ion accumulation in root tissues (periderm, xylem and phloem tissues) and in leaves of different ages was assessed for carrot plants grown in the field with a low level of salinity (5.8 mM Na(+) and 7.5 mM Cl(-)) and in a glasshouse with salinity ranging from 1-80 mM. At low levels of salinity (1-7.5 mM), in both the field and glasshouse, carrot leaves accumulated high concentrations of Cl(-) (140-200 mM); these appear to be the result of a high affinity for Cl(-) uptake and a low retention of Cl(-) in the root system. However, Cl(-) uptake is under tight control, with an 80-fold increase in external salinity resulting in only a 1.5-fold change in the Cl(-) concentration of the shoot and no increase in the Cl(-) concentration of the root xylem tissue. In contrast to Cl(-), shoot Na(+) concentrations were comparatively low (30-40 mM) but increased by seven-fold when salinity was increased by 80-fold. Growth over the 56-d treatment period in the glasshouse was insensitive to salinity less than 20 mM, but at higher concentrations the yield of carrot tap roots declined by 7 % for each 10 mM increase in salinity. At low levels of salinity the accumulation of high concentrations of Cl(-) (150 mM) in carrot laminae did not appear to limit leaf gas exchange. However, photosynthesis and stomatal conductance were reduced by 38 and 53 %, respectively, for plants grown at a salinity of 80 mM compared with those grown at 1 mM. Salinity-induced reductions in both p(i) and carbon isotope discrimination (delta) were small (2.5 Pa and 1.4 per thousand, respectively, at 80 mM) indicating that the reduction in photosynthesis was only marginally influenced by CO(2) supply. At a salinity of 80 mM the photosynthetic capacity was reduced, with a 30 % reduction in the CO(2)-saturated rate of photosynthesis (A(max)) and a 40 % reduction in both the apparent rate of RuBP-carboxylase-limited CO(2) fixation (V(cmax)) and the electron transport rate limiting RuBP regeneration (J(max)). This study has shown that carrot growth and leaf gas exchange are insensitive to the high leaf Cl(-) concentrations that occur at low levels (1-7 mM) of salinity. However, growth is limited at salinity levels above 20 mM and leaf gas exchange is limited at salinity levels above 8 mM.

Molecular and pharmacological characterization of GABA(A) receptor alpha1 subunit knockout mice.

GABA(A) receptors mediate fast inhibitory neurotransmission in the central nervous system (CNS), and approximately half of these receptors contain alpha1 subunits. GABA(A) receptor alpha1 subunits are important for receptor assembly and specific pharmacological responses to benzodiazepines. Plasticity in GABA(A) receptor alpha1 subunit expression is associated with changes in CNS excitability observed during normal brain development, in animal models of epilepsy, and upon withdrawal from alcohol and benzodiazepines. To examine the role of alpha1 subunit-containing GABA(A) receptors in vivo, we characterized receptor subunit expression and pharmacological properties in cerebral cortex of knockout mice with a targeted deletion of the alpha1 subunit. The mice are viable but exhibit an intention tremor. Western blot analysis confirms the complete loss of alpha1 subunit peptide expression. Stable adaptations in the expression of several GABA(A) receptor subunits are observed in the fifth to seventh generations, including decreased expression of beta2/3 and gamma2 subunits and increased expression of alpha2 and alpha3 subunits. There was no change in alpha4, alpha5, or delta subunit peptide levels in cerebral cortex. Knockout mice exhibit loss of over half of GABA(A) receptors measured by [(3)H]muscimol, [(3)H]2-(3-carboxyl)-3-amino-6-(4-methoxyphenyl)-pyridazinium bromide ([(3)H]SR-95531), and t-butylbicyclophosphoro[(35)S]thionate ([(35)S]TBPS) binding. [(3)H]Ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate ([(3)H]Ro15-4513) binding is reduced by variable amounts in different regions across brain. GABA(A) receptor alpha1(-/-) mice lose all high-affinity [(3)H]zolpidem binding and about half of [(3)H]flunitrazepam binding in the cerebral cortex. The potency and maximal efficacy of muscimol-stimulated (36)Cl(-) uptake in cerebral cortical synaptoneurosomes are reduced in alpha1(-/-) mice. Furthermore, knockout mice exhibit increased bicuculline-induced seizure susceptibility compared with wild-type mice. These data emphasize the significance of alpha1 subunit expression and its involvement in the regulation of CNS excitability.

Proton pump activity is required for active uptake of chloride in isolated amphibian skin exposed to freshwater.

Net proton secretion and unidirectional chloride fluxes were measured in isolated skin of toads ( Bufo bufo) and frogs ( Rana esculenta) mounted in an Ussing chamber and exposed to a Ringer's solution on the serosal side and a freshwater-like solution (1-3 mM Cl(-)) on the external side. Active proton secretion was 34.2+/-2.0 pmol.cm(-2).s(-1) ( n=18) in frog skin, and 16.7+/-1.7 pmol.cm(-2).s(-1) ( n=10) in toad skin. Proton secretion by toad skin was dependent on the transepithelial potential ( V(T)), and an amiloride-insensitive short-circuit current was stimulated by exogenous CO(2)/HCO(3)(-), indicating the presence of a rheogenic proton pump. Cl(-) influx was 37.4+/-7.5 pmol.cm(-2).s(-1) ( n=14) in frog skin and 19.5+/-3.5 pmol.cm(-2).s(-1) ( n=11) in toad skin. In toad skin, the mean Cl(-) flux ratio was larger than expected for simple electro-diffusion. In 8 of 11 sets of paired skins, influx was greater than the efflux indicating active uptake of Cl(-). Cl(-) influx in toad skin was unaffected by large perturbations (100-150 mV) of V(T), which was accomplished by adding amiloride to the outer bath under open circuit conditions. A component of the Cl(-) efflux seemed to be dependent on V(T). 4,4'-Diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS; 0.3 mM or 1.3 mM) inhibited Cl(-) influx and, surprisingly, increased Cl(-) efflux in toad skin. Influx and efflux of Cl(-) in toad skin were highly dependent on the external [Cl(-)] in the freshwater range (0.1-4 mM). (36)Cl(-) influx decreased whereas the total Cl(-) efflux increased as a function of external [Cl(-)]. These data indicate the presence of a DIDS-sensitive, electroneutral carrier mechanism with an external binding site for Cl(-). Ethoxzolamide (100 micro M), an inhibitor of carbonic anhydrase, reduced proton secretion and Cl(-) influx in frog skin. Concanamycin A (0.1-10 micro M), a specific vacuolar-type proton pump (V-ATPase) inhibitor, significantly reduced proton secretion in frog skin. In addition, concanamycin A (1 micro M) significantly reduced Cl(-) influx in frog skin. We suggest that the active proton secretion and Cl(-) influx are coupled. We hypothesise that an apical V-ATPase is capable of energising active Cl(-) uptake in fresh water by creating a favourable gradient for an apical HCO(3)(-) exit in exchange for external Cl(-). The data also suggest that a carbonic anhydrase activity provides H(+) and HCO(3)(-) for apically co-expressed proton pumps and Cl(-)/HCO(3)(-) exchangers.

The characterization of ion regulation in Amazonian mosquito larvae: evidence of phenotypic plasticity, population-based disparity, and novel mechanisms of ion uptake.

This study is the first step in characterizing ion uptake mechanisms of mosquito larvae from the Amazon region of Brazil. Hemolymph NaCl levels and rates of unidirectional Na(+) and Cl(-) uptake were measured in larvae of Aedes aegypti and Culex quinquefasciatus in a series of environmental manipulations that are known to challenge ion regulation in other aquatic animals. Despite being reared for numerous generations in dilute media (20 micromol L(-1) NaCl), both species were able to maintain high hemolymph NaCl concentrations, a departure from previous studies. Exposure to distilled water or high-NaCl media did not affect hemolymph ion levels, but pH 3 caused significant decreases in hemolymph Na(+) and Cl(-) levels in both species. Exposure to water from Rio Negro (pH 5.5), an organically rich but ion-poor body of water, did not disturb hemolymph Na(+) and Cl(-) levels or the uptake of these ions. Acute exposure to control media or Rio Negro water titrated to pH 3.5 caused inhibition of Na(+) uptake and stimulation of Cl(-) uptake in C. quinquefasciatus, but A. aegypti larvae experienced only a significant reduction of Na(+) uptake in Rio Negro/pH 3.5 treatment. The stimulation of Cl(-) uptake at low pH has been documented only in aquatic insects and differs from all other invertebrate and vertebrate species. A similar pattern of Na(+) uptake inhibition and Cl(-) uptake stimulation was observed in A. aegypti larvae exposed to bafilomycin A(1), a blocker of V-type H(+) ATPase. Culex quinquefasciatus larvae were unaffected by this drug. Both Na(+) and Cl(-) uptake were reduced when C. quinquefasciatus larvae were exposed to acetazolamide, indicating that H(+) and HCO(3)(-), derived from hydration of CO(2), are involved with Na(+) and Cl(-) uptake. Kinetic analysis of Na(+) and Cl(-) uptake in C. quinquefasciatus, A. aegypti, and Anopheles nuneztovari larvae indicate that these Amazonian species share similar high-capacity and high-affinity mechanisms. Comparison of the Amazonian C. quinquefasciatus with a Californian population provided evidence of both phenotypic plasticity and population disparity in Na(+) and Cl(-) uptake, respectively. When the California population of C. quinquefasciatus was reared in a medium similar to that of the Amazonian group (60 micromol L(-1) NaCl) instead of 4,000 micromol L(-1) NaCl, larvae increased both Na(+) uptake capacity (J(max)) and affinity (i.e., reduced K(m)), yet Cl(-) uptake did not change from its nonsaturating, low-capacity pattern. In the reverse experiment, Amazonian C. quinquefasciatus demonstrated plasticity in both Na(+) and Cl(-) uptake by significantly reducing rates when held in 4,000 micromol L(-1) NaCl for 3 d.

Ion regulatory patterns of mosquito larvae collected from breeding sites in the Amazon rain forest.

We examined the ion composition of mosquito breeding sites located in the Amazon rain forest and the ion regulatory patterns of larvae from these habitats. We found larvae of Toxorhynchites haemorroidalis, Limatus durhamii, Culex (Carrollia) bonnei, and Culex (Culex) sp. residing in fallen palm bracts, leaves, and tree holes that were filled with water. These breeding sites had micromolar levels of Na(+) (1.6-99 micromol L(-1)), but K(+) and Cl(-) concentrations were higher and varied over a large range (231-17,615 micromol L(-1) K(+); 355-2,700 micromol L(-1) Cl(-)). Despite the variability in environmental ion levels and ratios, all four species maintain high hemolymph NaCl levels (80-120 mmol L(-1) Na(+); 60-80 mmol L(-1) Cl(-)). However, the species differed in the means by which they maintain hemolymph ion balance, as indicated by the range of unidirectional Na(+) and Cl(-) uptake rates. Toxorhynchites haemorroidalis had extremely low rates of Na(+) uptake and undetectable Cl(-) uptake, whereas L. durhamii had high rates of uptake for both ions. This variability in rates of uptake may reflect species differences in rates of diffusive ion loss (i.e., permeability). We observed the same curious pattern of Na(+) inhibition and Cl(-) stimulation by low-pH exposure in all four species of mosquitoes, as has been documented in other mosquitoes and aquatic insects. Kinetic analyses of Na(+) and Cl(-) uptake in C. bonnei larvae revealed an unusual pattern of Na(+) uptake that increases linearly (nonsaturable) to extremely high rates, while Cl(-) uptake is a low-affinity, low-capacity system. This pattern contrasts with L. durhamii and Culex (Culex) sp. larvae, which had large increases in both Na(+) and Cl(-) uptake when external NaCl levels were increased. Our results suggest that although these rain forest mosquito larvae are residing in habitats with similar low Na(+), high Cl(-) composition and maintain similar hemolymph NaCl levels, the underlying mechanisms of ion regulation differ among the species.

Surface termination dependence of the reactivity of single crystal hematite with CCl4

We describe ultrahigh vacuum Auger electron spectrometric measurements of the uptake of chlorine following the room temperature exposure of single crystal hematite, α-Fe2O3, to CCl4. We compare the surface chemistry of two specific surface phases formed on the basal plane of α-Fe2O3: the Fe3O4(111)-(2×2) “selvedge” and the α-Fe2O3/Fe1−xO “biphase.” For Fe3O4(111)-(2×2) an estimated saturation level of Cl of ∼75% of a monolayer is readily attained. Carbon uptake is well below that expected for simple stoichiometric dissociative chemisorption, consistent with desorption of organic products during the surface reaction. Low energy electron diffraction measurements suggest that, dependent upon preparation procedures, at least two types of α-Fe2O3/Fe1−xO biphase structures can be formed. Surprisingly, upon exposure to CCl4, Cl uptake does not occur on either of these biphase surfaces, despite the fact that these surfaces are thought to have the same surface concentrations of iron and oxygen as Fe3O4(111). The dramatic difference between the reactivity of the Fe3O4 and biphase surfaces suggests that the active site for the dissociative adsorption of CCl4 on Fe3O4(111)-(2×2) comprises both an iron cation and an oxygen anion with a surface-normal-oriented dangling bond that is uncapped by iron cations. Electron stimulated and thermal desorption of Cl from the saturated Fe3O4(111)-(2×2) selvedge is also reported.

NaCl and fluid secretion by the intestine of the teleost Fundulus heteroclitus: involvement of CFTR.

Sections of posterior intestine of the euryhaline killifish Fundulus heteroclitus adapted to sea water were stimulated by the calcium ionophore ionomycin (1 micromol l(-1)) in combination with agents to elevate intracellular cyclic AMP levels, 0.5 mmol l(-1) dibutyryl-cyclic AMP (db-cAMP) with 0.1 mmol l(-1) 3-isobutyl-1-methylxanthine (IBMX). Intestinal bag preparations from recently fed animals (but not from overnight unfed animals) changed from fluid absorption (+18.9+/-8.30 microl cm(-2) h(-1), N=8) in the untreated control period to net fluid secretion after stimulation (-7.43+/-1.30 microl cm(-2) h(-1), N=8, P<0.01; means +/- S.E.M.), indicative of the capacity of teleost intestine to undergo secretion. Posterior intestinal pieces mounted in vitro in Ussing-style membrane chambers showed net Cl(-) uptake (+2.245+/-0.633 microequiv cm(-2) h(-1), N=7) that turned to net secretion following stimulation by ionomycin + db-cAMP + IBMX (-3.809+/-1.22 microequiv cm(-2) h(-1), N=7, P<0.01). Mucosal application of the anion channel blocker 1 mmol l(-1) diphenylamine-2-carboxylate (DPC) after ionomycin + db-cAMP + IBMX treatment significantly reduced serosal-to-mucosal unidirectional Cl(-) flux (P<0.001), net Cl(-) flux (P<0.05), short-circuit current (I(sc), P<0.001) and tissue conductance (G(t), P<0.001), while 0.1 mmol l(-1) 4,4'-diisothiocyano-2,2'-stilbene-disulphonic acid (DIDS, a blocker of anion exchange) was without effect. Stimulation by db-cAMP + IBMX (no ionomycin) significantly increased unidirectional fluxes, I(sc) and G(t) but did not produce net Cl(-) secretion. Ionomycin alone produced a transient increase in I(sc) but had no effect on G(t) and caused no significant changes in unidirectional or net Cl(-) fluxes. Addition of db-cAMP + IBMX after ionomycin treatment produced net secretion of Cl(-) and large increases in unidirectional fluxes and G(t). Cystic fibrosis transmembrane conductance regulator (CFTR) was immunocytochemically localized with a monoclonal mouse antibody to the carboxy terminus and found to be present in the cytoplasm and basolateral membranes of all enterocytes and in the brush-border membrane of some cells, whereas NKCC immunofluorescence, demonstrating the presence of the Na(+)/K(+)/2Cl(-) cotransporter, was present in the cytoplasm and brush-border membrane. We conclude that the teleost intestine is capable of salt and fluid secretion only if intracellular Ca(2+) and cyclic AMP pathways are stimulated together and that this secretion appears to involve activation of CFTR ion channels in the apical membrane of a subpopulation of enterocytes.