Effects of ouabain on chloride movements across the seawater eel intestine

Summary Simultaneous measurements of transepithelial potential difference (PD) and net water flux were made in the stripped intestine of seawater eels, and the effects of ouabain on these two parameters were examined in normal Ringer solution or under a chloride concentration gradient. Ouabain reduced the serosa-negative PD and the net water flux in normal Ringer solution with a linear relationship between the PD and the net water flux. Removal of K⁺ from the Ringer solution on both serosal and mucosal sides also reduced the PD and the net water flux to approximately zero. On the other hand, blocking the Na⁺–K⁺ pump by ouabain, K⁺-free or Na⁺-free Ringer solution increased the diffusion potential for Cl^–. Inhibition of Cl^– transport and increment in Cl^– permeability by ouabain occurred almost simultaneously. It is likely, therefore, that Cl^– transport as well as Cl^– permeability is dependent on Na⁺–K⁺ pump activity. A possible mechanism of dependence of Cl^– transport on the Na⁺–K⁺ pump is discussed in relation to the increment in Cl^– permeability.     

Na+-coupled Cl− transport in the gastric mucosa of the guinea pig

The Cl^–/HCO ₃ ^– exchange mechanism usually postulated to occur in gastric mucosa cannot account for the Na⁺-dependent electrogenic serosal to mucosal Cl^– transport often observed. It was recently suggested that an additional Cl^– transport mechanism driven by the Na⁺ electrochemical potential gradient may be present on the serosal side of the tissue. To verify this, we have studied Cl^– transport in guinea pig gastric mucosa. Inhibiting the (Na⁺, K⁺) ATPase either by serosal addition of ouabain or by establishing K⁺-free mucosal and serosal conditions abolished net Cl^– transport. Depolarizing the cell membrane potential with triphenylmethylphosphonium (a lipid-soluble cation), and hence reducing both the Na⁺ and Cl^– electrochemical potential gradients, resulted in inhibition of net Cl^– flux. Reduction of short-circuit current on replacing Na⁺ by choline in the serosal bathing solution was shown to be due to inhibition of Cl^– transport. Serosal addition of diisothiocyanodisulfonic acid stilbene (an inhibitor of anion transport systems) abolished net Cl^– flux but not net Na⁺ flux. These results are compatible with the proposed model of a Cl^–/Na⁺ cotransport mechanism governing serosal Cl^– entry into the secreting cells. We suggest that the same mechanism may well facilitate both coupled Cl^–/Na⁺ entry and coupled HCO ₃ ^– /Na⁺ exit on the serosal side of the tissue.     

Ca2+ transport by opercular epithelium of the fresh water adapted euryhaline teleost,Fundulus heteroclitus

We examined transepithelial transport of Ca²⁺ across the isolated opercular epithelium of the euryhaline killifish adapted to fresh water. The opercular epithelium, mounted in vitro with saline on the serosal side and fresh water (0.1 mmol·l^–1 Ca²⁺) bathing the mucosal side, actively transported Ca²⁺ in the uptake direction; net flux averaged 20–30 nmol·cm^–2·h^–1. The rate of Ca²⁺ uptake varied linearly with the density of mitochondria-rich cells in the preparations. Ca²⁺ uptake was saturable, apparent K _1/2 of 0.348 mmol·l^–1, indicative of a multistep transcellular pathway. Ca²⁺ uptake was inhibited partially by apically added 0.1 mmol·l^–1 La³⁺ and 1.0 mmol·l^–1 Mg²⁺. Addition of dibutyryl-cyclic adenosine monophosphate (0.5 mmol·l^–1)+0.1 mmol·l^–1 3-isobutyl-l-methylxanthine inhibited Ca²⁺ uptake by 54%, but epinephrine, clonidine and isoproterenol were without effect. Agents that increase intracellular Ca²⁺, thapsigargin (1.0 mol·l^–1, serosal side), ionomycin (1.0 mol·l^–1, serosal side) and the calmodulin blocker trifluoperazine (50 mol·l^–1, mucosal side) all partially inhibited Ca²⁺ uptake. In contrast, apically added ionomycin increased mucosal to serosal unidirectional Ca²⁺ flux, indicating Ca²⁺ entry across the apical membrane is rate limiting in the transport. Verapamil (10–100 mol·l^–1, mucosal side), a Ca²⁺ channel blocker, had no effect. Results are consistent with a model of Ca²⁺ uptake by mitochondria rich cells that involves passive Ca²⁺ entry across the apical membrane via verapamil-insensitive Ca²⁺ channels, intracellular complexing of Ca²⁺ by calmodulin and basolateral exit via an active transport process. Increases in intracellular Ca²⁺ invoke a downregulation of transcellular Ca²⁺ transport, implicating Ca²⁺ as a homeostatic mediator of its own transport.Abbreviations DASPEI 2-(4-dimethylaminostyryl)-N-ethylpyridinium iodide - db-cAMP dibutyryl-cyclic adenosine monophosphate - FW fresh water - G _t transepithelial conductance - I _sc short-circuit current - IBMX 3-isobutyl-1-methylxanthine - SW sea water - TFP trifluoperazine - V _t transepithelial potential     

Pathways for bicarbonate transfer across the serosal membrane of turtle urinary bladder: Studies with a disulfonic stilbene

Summary Bicarbonate is transferred across the serosal (S) membrane of the epithelial cells of the turtle bladder in two directions. Cellular HCO ₃ ^– generated behind the H⁺ pump moves across this membrane into the serosal solution. This efflux of HCO ₃ ^– is inhibited by SITS (4-isothiocyano-4-acetamido-2,2-disulfonic stilbene). When HCO ₃ ^– is added to the serosal solution it is transported across the epithelium in exchange for absorbed Cl^–. This secretory HCO ₃ ^– flow traverses the serosal cell membrane in the opposite direction. In this study the effects of serosal addition of 5×10^–4 m SITS on HCO ₃ ^– secretion and Cl^– absorption were examined. The rate of H⁺ secretion was brought to zero by an opposing pH gradient, and 20mm HCO ₃ ^– was added toS. HCO ₃ ^– secretion, measured by pH stat titration, was equivalent to the increase inMS Cl^– flux after HCO ₃ ^– addition. Neither theSM flux of HCO ₃ ^– nor theMS flux of Cl^– were affected by SITS. In the absence of electrochemical gradients, net Cl^– absorption was observed only in the presence of HCO ₃ ^– in the media; under such conditions, unidirectional and net fluxes of Cl^– were not altered by serosal or mucosal SITS. H⁺ secretion, however, measured simultaneously as the short-circuit current in ouabain-treated bladders decreased markedly after serosal SITS. The inhibition of the efflux of HCO ₃ ^– in series with the H⁺ pump and the failure of SITS to affect HCO ₃ ^– secretion and Cl^– absorption suggest that the epithelium contains at least two types of transport systems for bicarbonate in the serosal membrane.     

Bicarbonate-dependent chloride absorption in small intestine: Ion fluxes and intracellular chloride activities

Summary Proximal, stripped segments of small intestine from the urodeleAmphiuma were short-circuited in media containing Na⁺, Cl^– and HCO ₃ ^– . Under these conditions there was a large net absorption of Cl^–, a small net absorption of Na⁺ and a residual flux (J _Net ^R ) consistent with HCO ₃ ^– secretion. Net Cl^– absorption correlated with the short-circuit current (I _sc); net Na⁺ absorption correlated negatively withJ _Net ^R . Acetazolamide eliminated theI _sc, lowered Cl^– absorption by 50%, and reduced net Na⁺ absorption without alteringJ _Net ^R . Benzolamide inhibited theI _sc without alteringJ _Net ^R . Benzolamide inhibited theI _sc more rapidly when applied on the mucosal surface. Replacement of Na⁺ or HCO ₃ ^– (and CO₂) in the media eliminated theI _sc, net Cl^– absorption and the residual flux. Likewise, inclusion of the stilbene SITS in the serosal media eliminated theI _sc, net Cl^– absorption and the residual flux. The cytoplasmic activity of Cl^– (a _ci ^a ) was determined with single and double-barreled microelectrodes. Thea _ci ^a of villus absorptive cells in normal media was 21.0mm and in excess of that expected on the basis of electrochemical equilibrium of Cl^– at the mucosal membrane. Active Cl^– accumulation was also observed in the presence of acetazolamide but was eliminated upon replacement of media Na⁺ with choline. The mucosal membrane potential was depolarized upon replacement of media Na⁺. It is concluded that Cl^– is actively absorbed into intestinal cells ofAmphiuma by an electrogenic process located in the mucosal membrane. Depending on the level of intracellular HCO ₃ ^– , accumulated Cl^– may diffuse passively back into the mucosal media or undergo exchange with bath HCO ₃ ^– at the serosal membrane.     

Electrogenic Cl− absorption byAmphiuma small intestine: Dependence on serosal Na+ from tracer and Cl− microelectrode studies

Summary The Na⁺ requirement for active, electrogenic Cl^– absorption byAmphiuma small intestine was studied by tracer techniques and double-barreled Cl^–-sensitive microelectrodes. Addition of Cl^– to a Cl^–-free medium bathingin vitro intestinal segments produced a saturable (K _m =5.4mm) increase in shortcircuit current (I _sc) which was inhibitable by 1mm SITS. The selectivity sequence for the anion-evoked current was Cl^–=Br^–>SCN^–>NO ₃ ^– >F^–=I^–. Current evoked by Cl^– reached a maximum with increasing medium Na concentration (K _m =12.4mm). Addition of Na⁺, as Na gluconate (10mm), to mucosal and serosal Na⁺-free media stimulated the Cl^– current and simultaneously increased the absorptive Cl^– flux (J _ms ^Cl ) and net flux (J _net ^Cl ) without changing the secretory Cl^– flux (J _sm ^Cl ). Addition of Na⁺ only to the serosal fluid stimulatedJ _ms ^Cl much more than Na⁺ addition only to the mucosal fluid in paired tissues. Serosal DIDS (1mm) blocked the stimulation. Serosal 10mm Tris gluconate or choline gluconate failed to stimulateJ _ms ^Cl . Intracellular Cl^– activity (a _Cl ⁱ ) in villus epithelial cells was above electrochemical equilibrium indicating active Cl^– uptake. Ouabain (1mm) eliminated Cl^– accumulation and reduced the mucosal membrane potential _m over 2 to 3 hr. In contrast, SITS had no effect on Cl^– accumulation and hyperpolarized the mucosal membrane. Replacement of serosal Na⁺ with choline eliminated Cl^– accumulation while replacement of mucosal Na⁺ had no effect. In conclusion by two independent methods active electrogenic Cl^– absorption depends on serosal rather than mucosal Na⁺. It is concluded that Cl^– enters the cell via a primary (rheogenic) transport mechanism. At the serosal membrane the Na⁺ gradient most likely energizes H⁺ export and regulates mucosal Cl^– accumulation perhaps by influencing cell pH or HCO ₃ ^– concentration.     

Relationship between coupled Na+−K+−Cl− transport and water absorption across the seawater eel intestine

Summary Simultaneous measurements of net ion and water fluxes were made in the stripped intestine of the seawater eel, and the relationship between Na⁺, K⁺, Cl^– and water transport were examined in the presence of mucosal KCl and serosal NaCl Ringer (standard condition). When Cl^– was removed from both sides of the intestine, net K⁺ flux from mucosa to serosa was reduced, accompanied by complete blockage of water absorption. Since it has been shown that net Cl^– and water fluxes depend on K⁺ transport under the standard condition (Ando 1983), the interdependence of K⁺ and Cl^– transport suggests the existence of a coupled KCl transport system, while the parallelism between the net Cl^– and water fluxes suggests that water absorption is linked to the coupled KCl transport. The coupled KCl and water transport were inhibited by treatment with ouabain or with Na⁺-free Ringer solutions, suggesting the existence of a Na⁺-dependent KCl transport system and linkage of water absorption to the coupled Na⁺–K⁺–Cl^– transport. Since ouabain blocked the active Na⁺–K⁺–Cl^– transport almost completely, the permeability coefficients for K⁺ and Na⁺ through the paracellular shunt pathway were estimated as P_K=0.076 and P_Na=0.058 cm/h, and P_Cl was calculated as 0.005 cm/h. Although Na⁺-independent K⁺ and Cl^tt- fluxes were observed again in the present study, these fluxes were not inhibited by CN^–, ouabain or diuretics, and evoked even after blocking the Na⁺–K⁺–Cl^– transport completely with ouabain. These results indicate that the Na⁺-independent K⁺ and Cl^– fluxes are distinct from the active Na⁺–K⁺–Cl^– transport and are not themselves active.     

Modes of Cl− transport across the mucosal and serosal membranes of urodele intestinal cells

Summary The characteristics of Cl^– movement across luminal and basolateral membranes ofAmphiuma intestinal absorptive cells were studied using Cl^–-sensitive microelectrodes and tracer³⁶Cl techniques. Intracellular Cl^– activity (a _Cl ⁱ ) was unchanged when serosal Cl^– was replaced; when luminal Cl^– was replaced cell Cl^– was rapidly lost. Accordingly, the steady statea _Cl ⁱ could be varied by changing the luminal [Cl]. As luminal [Cl] was raised from 1 to 86mM,a _Cl ⁱ rose in a linear manner, the mucosal membrane hyperpolarized, and the transepithelial voltage became serosa negative. In contrast, the rate of Cl^– transport from the cell into the serosal medium, measured as the SITS-inhibitable portion of the Cl^– absorptive flux, attained a maximum whena _Cl ⁱ reached an apparent value of 17mm, indicating the presence of a saturable, serosal transport step. The stilbeneinsensitive absorptive flux was linear with luminal [Cl], suggestive of a paracellular route of movement. Intracellulara _Cl was near electrochemical equilibrium at all but the lowest values of luminal [Cl] after interference produced by other anions was taken into account.a _Cl ⁱ was unaffected by Na replacement, removal of medium K, or elevation of medium HCO ₃ ^– . Mucosae labeled with³⁶Cl lost isotope into both luminal and serosal media at the same rate and from compartments of equal capacity. Lowering luminal [Cl] or addition of theophylline enhanced luminal Cl^– efflux. It is concluded that a conductive Cl leak pathway is present in the luminal membrane. Serosal transfer is by a saturable, stilbene-inhibitable pathway. Luminal Cl^– entry appears to be passive, but an electrogenic uptake cannot be discounted.     

Separate,Ca2+-activated K+ and Cl− transport pathways in Ehrlich ascites tumor cells

Summary The net loss of KCl observed in Ehrlich ascites cells during regulatory volume decrease (RVD) following hypotonic exposure involves activation of separate conductive K⁺ and Cl^– transport pathways. RVD is accelerated when a parallel K⁺ transport pathway is provided by addition of gramicidin, indicating that the K⁺ conductance is rate limiting. Addition of ionophore A23187 plus Ca²⁺ also activates separate K⁺ and Cl^– transport pathways, resulting in a hyperpolarization of the cell membrane. A calculation shows that the K⁺ and Cl^– conductance is increased 14-and 10-fold, respectively. Gramicidin fails to accelerate the A23187-induced cell shrinkage, indicating that the Cl^– conductance is rate limiting. An A23187-induced activation of⁴²K and³⁶Cl tracer fluxes is directly demonstrated. RVD and the A23187-induced cell shrinkage both are: (i) inhibited by quinine which blocks the Ca²⁺-activated K⁺ channel. (ii) unaffected by substitution of NO ₃ ^– or SCN^– for Cl^–, and (iii) inhibited by the anti-calmodulin drug pimozide. When the K⁺ channel is blocked by quinine but bypassed by addition of gramicidin, the rate of cell shrinkage can be used to monitor the Cl^– conductance. The Cl^– conductance is increased about 60-fold during RVD. The volume-induced activation of the Cl^– transport pathway is transient, with inactivation within about 10 min. The activation induced by ionophore A23187 in Ca²⁺-free media (probably by release of Ca²⁺ from internal stores) is also transient, whereas the activation is persistent in Ca²⁺-containing media. In the latter case, addition of excess EGTA is followed by inactivation of the Cl^– transport pathway. These findings suggest that a transient increase in free cytosolic Ca²⁺ may account for the transient activation of the Cl^– transport pathway. The activated anion transport pathway is unselective, carrying both Cl^–, Br^–, NO ₃ ^– , and SCN^–. The anti-calmodulin drug pimozide blocks the volume- or A23187-induced Cl^– transport pathway and also blocks the activation of the K⁺ transport pathway. This is demonstrated directly by⁴²K flux experiments and indirectly in media where the dominating anion (SCN^–) has a high ground permeability. A comparison of the A23187-induced K⁺ conductance estimated from⁴²K flux measurements at high external K⁺, and from net K^– flux measurements suggests single-file behavior of the Ca²⁺-activated K⁺ channel. The number of Ca²⁺-activated K⁺ channels is estimated at about 100 per cell.     

Intestinal HCO 3 − secretion inAmphiuma: Stimulation by mucosal Cl− and serosal Na+

Summary The requirement for Na⁺ and Cl^– in the bathing media to obtain a maximal HCO ₃ ^– secretory flux ( ) across isolated short-circuitedAmphiuma duodenum was investigated using titration techniques and ion substitution. Upon substitution of media Na⁺ with choline, HCO ₃ ^– secretion was markedly reduced. Replacement of media Cl^– produced a smaller reduction of . The presence of Cl^– enhanced HCO ₃ ^– secretion only if Na⁺ was also in the media. Elevation of media Na⁺ or Cl^– in the presence of the other ion produced a saturable increase of . In the presence of Na⁺, Cl^– stimulated when added to the mucosal but not the serosal medium. In the presence of Cl^–, Na⁺ elevated when added to the serosal but not the mucosal medium. The ability of mucosal Cl^– to stimulate was not apparently dependent on mucosal Na⁺. Simultaneous addition of 10mm Cl^– to the Na⁺-free mucosal medium and 10mm Na⁺ to the Cl^–-free serosal medium stimulated above levels produced by serosal Na⁺ alone. In conclusion, intestinal HCO ₃ ^– secretion required mucosal Cl^– and serosal Na⁺ and did not involve mucosal NaCl cotransport. The results are consistent with a mucosal Cl^– absorptive mechanism in series with parallel basolateral Na⁺–H⁺ and Cl^––HCO ₃ ^– exchange mechanisms.     

Chloride and potassium channels in U937 human monocytes

Summary Ionic channels in a human monocyte cell line (U937) were studied with the inside-out patch-clamp technique. A Ca²⁺-activated K⁺ channel and three Cl^–-selective channels were observed. The Ca²⁺-activated K⁺ channel had an inward-rectifying current-voltage relationship with slope conductance of 28 pS, and was not dependent on membrane potential. Among the three Cl^– channels, and outward-rectifying 28-pS channel was most frequently observed. The permeability ratio (Cl^–/Na⁺) was 4–5 and CH₃SO ₄ ^– was also permeant. The channel became less active with increasing polarizations in either direction, and was inactive beyond ±120 mV. The channel, observed as bursts, occasionally had rapid events within the bursts, suggesting the presence of another mode of kinetics. Diisothiocyanatostilbene-disulfonic acid (DIDS) blocked the channel reversibly in a dose-dependent manner. The second 328-pS Cl^– channel had a linear currentvoltage relationship and permeability ratio (Cl^–/Na⁺) of 5–6. This channel became less active with increasing polarizations and inactive beyond ±50 mV. DIDS blocked the channel irreversibly. The channel had multiple subconductance states. The third 15-pS Cl^– channel was least frequently observed and least voltage sensitive among the Cl^– channels. Intracellular Ca²⁺ or pH affected none of the three Cl^– channels. All three Cl^– channels had a latent period before being observed, suggesting inhibitory factor(s) presentin situ. Activation of the cells with interferon-, interferon-A or 12-O-tetradecanoylphorbol-13-acetate (TPA) caused no change in the properties on any of the channels.     

Dynamics of dissolved O2, CO2, CH4, and N2O in a tropical coastal swamp in southern Thailand

We studied the distribution of dissolved O₂, CO₂, CH₄, and N₂O in a coastal swamp system in Thailand with the goal to characterize the dynamics of these gases within the system. The gas concentrations varied spatially and seasonally in both surface and ground waters. The entire system was a strong sourcefor CO₂ and CH₄, and a possible sink for atmospheric N₂O. Seasonal variation in precipitation primarily regulated the redox conditions in the system. However, distributions of CO₂, CH₄, and N₂O in the river that received swamp waters were not always in agreement with redox conditions indicated by dissolvedO₂ concentrations. Sulfate production through pyriteoxidation occurred in the swamp with thin peat layerunder aerobic conditions and was reflected by elevatedSO ₄ ^2– /Cl^– in the river water. When SO ₄ ^2– /Cl^– was high, CO₂ and CH₄ concentrations decreased, whereas the N₂O concentration increased. The excess SO ₄ ^2– in the river water was thus identified as a potential indicator for gas dynamics in this coastal swamp system.     

Solution chemistry profiles of mixed-conifer forests before and after fire

Solution chemistry profiles of mixed-conifer forests in granitic catchments of the Sierra Nevada were measured for three years before (1987–1990) and three years after (1990–1993) prescribed fire. Wet deposition, throughfall and soil solution samplers were installed in both white-fir and giant-sequoia dominated forest stands underlain by poorly developed inceptisols. Stream water chemistry was monitored as part of an ongoing study of catchment outputs. Calcium, NO ₃ ^– and Cl^– were the major ions in precipitation. Canopy leaching increased mean concentrations of all major ions, especially K⁺ and Ca²⁺. Water flux through the soil occurred largely during spring snowmelt. Forest floor leachate represented the most concentrated solutions of major ions. Interaction with the mineral soil decreased mean concentrations of most species and the average composition of soil solutions closely resembled stream water at baseflow. Bicarbonate alkalinity, Ca²⁺, Mg²⁺, and Na⁺ were enriched in stream water relative to precipitation whereas inputs of H⁺, NH ₄ ⁺ , NO ₃ ^– and SO ₄ ^2– were retained within the catchments.Burning of the forest understory and litter layer increased solute concentrations in soil solution and stream water. Mean soil solution Ca²⁺, Mg²⁺ and K⁺ concentrations increased more than 10 fold, but the relative predominance of these cations was not affected by burning. Sulfate concentration, which was very low in soil solutions of undisturbed stands (<25 mmol_c m^–3), increased more than 100 times following fire. Ammonium concentration exhibited a rapid, short-term increase and then a decrease below pre-burn levels. Changes in soil solution chemistry were reflected in catchment outputs.Corresponding author.     

The effects of liming an Adirondack lake watershed on downstream water chemistry

Calcite treatment of chronically acidic lakes has improved fish habitat, but the effects on downstream water quality have not previously been examined. In this study, the spatial and temporal effects of watershed CaCO₃ treatment on the chemistry of a lake outlet stream in the Adirondack Mountains of New York were examined. Before CaCO₃ treatment, the stream was chronically acidic. During spring snowmelt before treatment, pH and acid-neutralizing capacity (ANC) in the outlet stream declined, and NO ₃ ^– and inorganic monomeric aluminum (Al_IM) concentrations increased sharply. During that summer, SO ₄ ^– and NO ₃ ^– concentrations decreased downstream, and dissolved organic carbon (DOC) concentrations and ANC increased, in association with the seasonal increase in decomposition of organic matter and the attendant SO ₄ ^– -reduction process. A charge-balance ANC calculation closely matched measured downstream changes in ANC in the summer and indicated that SO ₄ ^– reduction was the major process contributing to summer increases in ANC. Increases in Ca²⁺ concentration and ANC began immediately after CaCO₃ application, and within 3 months, exceeded their pretreatment values by more than 130 eq/L. Within 2 months after treatment, downstream decreases in Ca²⁺ concentration, ANC, and pH, were noted. Stream mass balances between the lake and the sampling site 1.5 km downstream revealed that the transport of all chemical constituents was dominated by conservative mixing with tributaries and ground water; however, non-conservative processes resulted in significant Ca²⁺ losses during the 13-month period after CaCO₃ treatment. Comparison of substrate samples from the buffered outlet stream with those from its untreated tributaries showed that the percentage of cation-exchange sites occupied by Ca²⁺ as well as non-exchangeable Ca, were higher in the outlet-stream substrate than in tributary-stream substrate. Mass-balance data for Ca²⁺ H⁺, Al_IM, and DOC revealed net downstream losses of these constituents and indicated that a reasonable set of hypothesized reactions involving Al_IM, HCO ₃ ^– , Ca²⁺, SO ₄ ^– NO ₃ ^– , and DOC could have caused the measured changes in stream acid/base chemistry. In the summer, the sharp decrease in ANC continued despite significant downstream decreases in SO₄ ^2– concentrations. After CaCO₃ treatment, reduction of SO ₄ ^– was only a minor contributor to ANC changes relative to those caused by Ca²⁺ dilution from acidic tributaries and acidic ground water, and Ca²⁺ interactions with stream substrate.     

Effects of calcium on growth and leaf ion concentrations of Gossypium hirsutum grown in saline hydroponic culture

The optimum Ca²⁺ concentration for growth of cotton (Gossypium hirsutum cv. Acala SJ-2) was in the range 1 to 15 mol m^–3 for plants growing in hydroponic culture with 100–150 mol m^–3 NaCl. Most saline (but not sodic) soils contain higher Ca²⁺ concentrations. CaCl₂ was inhibitory to the growth of cotton above 20–50 mol m^–3. Increasing concentrations of Ca²⁺ in the range 0–2 mol m^–2 drastically reduced Na⁺ accumulation in the leaves. As CaCl₂ concentrations were increased above the optimum for growth there was a further reduction in leaf Na⁺ accumulation, but this was more than offset by increased leaf Ca²⁺ and Cl^– concentrations. Leaf K⁺ concentrations were not much affected by changes in external CaCl₂ concentrations. The response of Mg²⁺ varied from an increase to a decrease with increasing external CaCl₂ and was influenced by nutritional status. There was no evidence that high Ca²⁺ caused a deficiency of Mg²⁺ in cotton. Except for Cl^–, whose concentrations tended to decrease initially and then increase as the CaCl₂ concentration increased, the anions were largely unaffected by changes in external CaCl₂.     

Calcium content of liming material and its effect on sulphur release in a coniferous forest soil

Soil columns with O + A (Experiment I) or Ohorizons (Experiment II) from a Haplic Podsol wereincubated at 15 °C for 368 and 29 + 106 days,respectively. Three types of liming material differingin Ca²⁺ content, i.e. calcium carbonate(CaCO₃), dolomite (CaMg(CO₃)₂) andmagnesium carbonate (MgCO₃), were mixed into theO horizons in equimolar amounts corresponding to 6000kg of CaCO₃ per ha. In the limed treatments ofExperiment I, the leaching of dissolved organic carbon(DOC) and the net sulphur mineralization (estimated asaccumulated SO ₄ ^2– leaching corrected forchanges in the soil pools of adsorbed and waterextractable SO ₄ ^2– ) increased with decreasingCa²⁺ content of the lime and increasing degree oflime dissolution. In relation to the controltreatment, only the MgCO₃ treatment resulted ina significantly higher net sulphur mineralization. InExperiment I the net sulphur mineralization was 4.06,1.68, 0.57, and 2.14 mg S in the MgCO₃,CaMg(CO₃)₂, CaCO₃ and control treatment,respectively. The accumulated SO ₄ ^2– leachingin Experiment II during the first 29 days was 1.70,0.74 and 0.48 mg S in the MgCO₃,CaMg(CO₃)₂ and control treatment,respectively. In the two experiments there wereconsistently significant positive correlations betweenleached amounts of SO ₄ ^2– and DOC. It wasconcluded that net sulphur mineralization was stronglyconnected to the solubilization of the organic matter(DOC formation) and that pH and/or Ca²⁺ ionsaffected the net sulphur mineralization through theireffects on organic matter solubility.     

Interaction between anions and the reduced folate/methotrexate transport system in L1210 cell plasma membrane vesicles: Directional symmetry and anion specificity for differential mobility of loaded and unloaded carrier

Summary The effect of various anions on the mediated influx and efflux of [³H]methotrexate by L1210 cell plasma membrane vesicles in a HEPES buffer system was studied. Our results show that flux is stimulated to the same extent in either direction when SO₄, P_i, or folate compounds (1,L5-CHO-folate-H₄, methotrexate), but not Cl^– was present in the opposite compartment. This implies the property of directional symmetry, a condition in which differential mobility of loaded and unloaded carriers occurs in both directions.We also observed a similarity in the specificity of the interaction between various anions and carrier in each orientation of the membrane, in the order, Cl^– P_i SO ₄ ^2– methotrexate < 1,L5-CHO-folate-H₄. Also, the absolute differential in mobility of loaded and unloaded carrier (assumed from the extent of transstimulation obtained) varied substantially among the anions examined. No stimulation was obtained with Cl^–, and stimulation was twofold with P_i, SO ₄ ^2– and methotrexate and fourfold with 1,L5-CHO-folate-H₄. Transstimulation of flux from either external or internal compartment only occurred when a positive gradient of total anions was maintained in the opposite compartment. Also, no stimulation occurred when the same equivalence of two different anions are present in opposing compartments. The concentration of anions required to transstimulate [³H]methotrexate influx was increased four- to 10-fold when vesicles were equilibrated in 145mM NaCl. These results suggest that under physiological conditions, concentrative uptake of methotrexate in intact L1210 cells as a result of anion exchange would require a large positive gradient in the total concentration of internalized anions.     

Ca2+ pump and Ca2+/H+ antiporter in plasma membrane vesicles isolated by aqueous two-phase partitioning from corn leaves

Summary Plasma membrane vesicles, which are mostly right side-out, were isolated from corn leaves by aqueous two-phase partitioning method. Characteristics of Ca²⁺ transport were investigated after preparing inside-out vesicles by Triton X-100 treatment.⁴⁵Ca²⁺ transport was assayed by membrane filtration technique. Results showed that Ca²⁺ transport into the plasma membrane vesicles was Mg-ATP dependent. The active Ca²⁺ transport system had a high affinity for Ca²⁺(K _m (Ca²⁺)=0.4 m) and ATP(K _m (ATP)=3.9 m), and showed pH optimum at 7.5. ATP-dependent Ca²⁺ uptake in the plasma membrane vesicles was stimulated in the presence of Cl^– or NO ₃ ^– . Quenching of quinacrine fluorescence showed that these anions also induced H⁺ transport into the vesicles. The Ca²⁺ uptake stimulated by Cl^– was dependent on the activity of H⁺ transport into the vesicles. However, carbonylcyanidem-chlorophenylhydrazone (CCCP) and VO ₄ ^3– which is known to inhibit the H⁺ pump associated with the plasma membrane, canceled almost all of the Cl^–-stimulated Ca²⁺ uptake. Furthermore, artificially imposed pH gradient (acid inside) caused Ca²⁺ uptake into the vesicles. These results suggest that the Cl^–-stimulated Ca²⁺ uptake is caused by the efflux of H⁺ from the vesicles by the operation of Ca²⁺/H⁺ antiport system in the plasma membrane. In Cl^–-free medium, H⁺ transport into the vesicles scarcely occurred and the addition of CCCP caused only a slight inhibition of the active Ca²⁺ uptake into the vesicles. These results suggest that two Ca²⁺ transport systems are operating in the plasma membrane from corn leaves, i.e., one is an ATP-dependent active Ca²⁺ transport system (Ca²⁺ pump) and the other is a Ca²⁺/H⁺ antiport system. Little difference in characteristics of Ca²⁺ transport was observed between the plasma membranes isolated from etiolated and green corn leaves.     

The absorption of calcium ions from the ovine reticulo-rumen

Net Ca²⁺ and Mg²⁺ absorption rates were measured in vivo from buffer solutions placed in the washed reticulo-rumen, isolated in situ in 30 conscious, trained sheep. An increase in concentration of short chain fatty acids (SCFA) in the buffer, over the range 0–50 mM, was shown to stimulate the net rates of absorption of Ca²⁺ and Mg²⁺ ions from the rumen. Similarly, the results of in vitro experiments, carried out with ovine rumen epithelium mounted in short-circuited Ussing chambers, showed that the absence of SCFA from the chamber fluid resulted in a reduction in J_net Ca²⁺ caused by reduced flux of Ca²⁺ ions in the mucosal to serosal direction (J_ms Ca²⁺). The addition of 1 mM acetazolamide, an inhibitor of carbonic anhydrase, to the ruminal buffer used in the in vivo experiments led to significant reductions in the net absorption rates of Ca²⁺and Mg²⁺ ions in the presence of SCFA (50 mmol l⁻¹) but not in the absence of SCFA. However, in the in vitro experiments, the addition of 60 μM ethoxyzolamide had no significant effect on J_net Ca²⁺. A reduction in pH of the intraruminal buffer in vivo from 6.8 to 5.4 led to significant increases in the net absorption rates of Ca²⁺and Mg²⁺ ions, an effect which was duplicated for Ca²⁺ in preliminary in vitro experiments in which the pH of the mucosal buffer was reduced from 7.4 to 5.4. This stimulatory effect was confined to J_ms Ca²⁺ and J_net Ca²⁺. Ussing chambers were also used to demonstrate that J_net Ca²⁺ was reduced by a high transmural potential difference (PD), caused by voltage clamping, independently of the mucosal K⁺ concentration. Both unidirectional Ca²⁺ fluxes consisted of a PD-dependent and a K⁺-insensitive PD-independent component. The latter may be represented by a Ca²⁺/2H⁺ antiporter. It is postulated that SCFA, and to a lesser extent H₂CO₃, can stimulate J_ms Ca²⁺ by activation of an apical Ca²⁺/2H⁺ antiporter through the provision of protons within the ruminal epithelial cell. A mild reduction in ruminal pH may also lead to a similar stimulation of this putative electroneutral exchange. Accepted: 26 July 2000     

Cyclic AMP-induced changes in membrane conductance ofNecturus gallbladder epithelial cells

Summary Enhanced cellular cAMP levels have been shown to increase apical membrane Cl^– and HCO ₃ ^– conductances in epithelia. We found that the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) increases cAMP levels inNecturus gallbladder. We used conventional open-tip and double-barreled Cl^–-selective microelectrodes to study the effects of IBMX on membrane conductances and intracellular Cl^– activities in gallbladders mounted in a divided chamber and bathed with Ringer's solutions at 23°C and pH 7.4. In HCO ₃ ^– -free media, 0.1mM IBMX added to the mucosal medium depolarized the apical membrane potentialV _a , decreased the fractional resistanceF _R , and significantly reduced intracellular Cl^– activity (a _Cl ⁱ ). Under control conditions,a _Cl ⁱ was above the value corresponding to passive distribution across the apical cell membrane. In media containing 25mM HCO ₃ ^– , IBMX caused a small transient hyperpolarization ofV _a followed by a depolarization not significantly different from that observed in HCO ₃ ^– -free Ringer's. Removal of mucosal Cl^–, Na⁺ or Ca²⁺ did not affect the IBMX-induced depolarization inV _a . The basolateral membrane ofNecturus gallbladder is highly K⁺ permeable. Increasing serosal K⁺ from 2.5 to 80mM, depolarizedV _a . Mucosal IBMX significantly reduced this depolarization. Addition of 10mM Ba²⁺, a K⁺ channel blocker, to the serosal medium depolarizedV _a and, essentially, blocked the depolarization induced by IBMX. These results indicate that mucosal IBMX increases apical HCO ₃ ^– conductance and decreases basolateral K⁺ conductance in gallbladder epithelial cells via a cAMP-dependent mechanism. The latter effect, not previously reported in epithelial tissues, appears to be the major determinant of the IBMX-induced depolarization ofV _a .