Modulation of the membrane potential of endothelial cells from the guinea pig aorta by intracellular alkalinization

Endothelium-dependent vasoactive substances are known to evoke complex changes in the endothelial membrane potential (MP) and to increase intracellular pH in endothelial cells (EC). In our present study, we investigated the effect of agents able to increase intracellular pH on the MP of intact guinea pig aortic EC, and also the effect of blocking of Na⁺−H⁺ exchanger on ATP-induced electrical responses. Intracellular alkalinization was induced either by addition of ammonium chloride (NH₄Cl) to the superfusate, or by changing the bath solution saturated with 10% CO₂+90% O₂ to a solution saturated with 100% O₂. Both approaches evoked hyperpolarization of EC. After intracellular Ca²⁺ chelation by pretreatment of aortic preparations with 20 μM BAPTA-AM, the amplitude of NH₄Cl-induced hyperpolarization dropped from 3.9±0.6 to 0.7±0.3 mV. After pretreatment with ATP, NH₄Cl-induced hyperpolarization was not abolished, whereas after caffeine pretreatment this hyperpolarization was not observed. In the Na⁺-free solution and in the presence of furosemide, ATP-evoked hyperpolarization became longer. The same effect was also observed in the presence of sodium acetate, which directly acidifies the cytosol. In the Ca²⁺-free solution, furosemide did not induce prolongation of ATP-evoked hyperpolarization. Taking into account the results, it could be proposed that, first, hyperpolarization of EC after intracellular alkalinization is a result of Ca²⁺ release from the intracellular stores sensitive both to an increase in intracellular pH and to caffeine application. Second, intracellular alkalinization, being a result of activation of Na⁺−H⁺-antiporter, inhibits influx of extracellular Ca²⁺ into EC under ATP stimulation.     

A Mathematical Model of the Pancreatic Ductal Epithelium

A mathematical model of the HCO⁻ ₃-secreting pancreatic ductal epithelium was developed using network thermodynamics. With a minimal set of assumptions, the model accurately reproduced the experimentally measured membrane potentials, voltage divider ratio, transepithelial resistance and short-circuit current of nonstimulated ducts that were microperfused and bathed with a CO₂/HCO⁻ ₃-free, HEPES-buffered solution, and also the intracellular pH of duct cells bathed in a CO₂/HCO⁻ ₃-buffered solution. The model also accurately simulated: (i) the effect of step changes in basolateral K⁺ concentration, and the effect of K⁺ channel blockers on basolateral membrane potential; (ii) the intracellular acidification caused by a Na⁺-free extracellular solution and the effect of amiloride on this acidification; and (iii) the intracellular alkalinization caused by a Cl⁻-free extracellular solution and the effect of DIDS on this alkalinization. In addition, the model predicted that the luminal Cl⁻ conductance plays a key role in controlling both the HCO⁻ ₃ secretory rate and intracellular pH during HCO⁻ ₃ secretion. We believe that the model will be helpful in the analysis of experimental data and improve our understanding of HCO⁻ ₃-transporting mechanisms in pancreatic duct cells. Received: 18 October 1995/Revised: 5 July 1996     

P2Y(2) receptor-mediated inhibition of amiloride-sensitive short circuit current in M-1 mouse cortical collecting duct cells

Extracellular nucleotides modulate renal ion transport. Our previous results in M-1 cortical collecting duct cells indicate that luminal and basolateral ATP via P2Y₂ receptors stimulate luminal Ca²⁺-activated Cl⁻ channels and inhibit Na⁺ transport. Here we address the mechanism of ATP-mediated inhibition of Na⁺ transport. M-1 cells had a transepithelial voltage (V _te ) of −31.4 ± 1.3 mV and a transepithelial resistance (R _te ) of 1151 ± 28 Ωcm². The amiloride-sensitive short circuit current (I _sc ) was −28.0 ± 1.1 μA/cm². The ATP-mediated activation of Cl⁻ channels was inhibited when cytosolic Ca²⁺ increases were blocked with cyclopiazonic acid (CPA). Without CPA the ATP-induced [Ca²⁺]_i increase was paralleled by a rapid and transient R _te decrease (297 ± 51 Ωcm²). In the presence of CPA, basolateral ATP led to an R _te increase by 144 ± 17 Ωcm² and decreased V _te from −31 ± 2.6 to −26.6 ± 2.5 mV. I _sc dropped from −28.6 ± 2.4 to −21.6 ± 1.9 μA/cm². Similar effects were observed with luminal ATP. In the presence of amiloride, ATP was without effect. This reflects ATP-mediated inhibition of Na⁺ absorption. Lowering [Ca²⁺]_i by removal of extracellular Ca²⁺ did not alter the ATP effect. PKC inhibition or activation were without effect. Na⁺ absorption was activated by pH_i alkalinization and inhibited by pH_i acidification. ATP slightly acidified M-1 cells by 0.05 ± 0.005 pH units, quantitatively not explaining the ATP-induced effect. In summary this indicates that extracellular ATP via luminal and basolateral P2Y₂ receptors inhibits Na⁺ absorption. This effect is not mediated via [Ca²⁺]_i, does not involve PKC and is to a small part mediated via intracellular acidification. Received: 9 February 2001/Revised: 17 May 2001     

Effect of bicarbonate,pH, methazolamide and stibenes on the intracellular potentials of cultured bovine corneal endothelial cells

Summary Micropuncture of cultured bovine corneal endothelial cells led to registrations stable for hours. Intracellular potentials were mainly in the range of –40 to –55 mV, average 46.3±0.6 mV (sem). Changes of extracellular [HCO ₃ ^– ] led to voltage transients, their amplitude depending logarithmically on [HCO ₃ ^– ] with a mean slope of 37.3±8.8 (sd) mV. After removal of bicarbonate/CO₂, a steady-state depolarization was seen. This steady-state depolarization, but not the voltage transients, could be reduced by 1mm Ba⁺⁺. After removal of bicarbonate, the voltage response to changes of extracellular potassium was reduced. Alteration of pH _i induced by permeable buffers (butyrate, glycodiazine and ammonium) also resulted in voltage transients, internal acidification being correlated with a hyperpolarization, and internal alkalinization with a depolarization. Also changes of external pH caused voltage responses, alkalinization causing a hyperpolarization, acidification a depolarization. Methazolamide, an inhibitor of carbonic anhydrase, as well as stilbenes (SITS or DIDS) caused a reduction of the voltage response to HCO ₃ ^– and pH. Their effects were additive. It is suggested that corneal endothelial cells possess one or two electrogenic transporters for HCO ₃ ^– or related species, one of which is inhibitable by stilbenes.     

Caffeine-induced electrical responses of intact endothelial cells

The mechanism of caffeine-induced endothelial-dependent relaxation of vascular smooth muscle cells has been studied by recording caffeine application-induced electrical responses from intact guinea pig aortic endothelial cells. Depending on the values of the membrane potential, caffeine evoked either hyperpolarizing responses (V _m<−45 mV, 88.9% of the cells tested), or depolarizing reactions (V _m>−45 mV). The mean amplitude of caffeine-induced hyperpolarization of endothelial cells was 11.2±5.5 mV, which is comparable with the amplitude of ATP-induced hyperpolarization. The amplitude of caffeine-induced depolarization was 8.9±3.4 mV, on average. It was shown that caffeine-induced hyperpolarization of endothelial cells is a result of calcium release from the intracellular stores with subsequent activation of calcium-dependent potassium channels. Intracellular calcium stores involved in caffeine-induced responses are different from those involved in ATP responses. It is concluded that calcium mobilization from the intracellular stores of endothelial cells and, possibly, activation of calcium entry contributes to the caffeine-induced endothelial-dependent relaxation of vascular smooth muscle cells.     

The role of external carbonic anhydrase in the photosynthetic use of inorganic carbon in the deep-water alga Phyllariopsis purpurascens (Laminariales, Phaeophyta)

Mechanisms of inorganic carbon assimilation were investigated in the deep-water alga Phyllariopsis purpurascens (C. Agardh) Henry et South (Laminariales, Phaeophyta). The gross photosynthetic rate as a function of external pH, at a constant concentration of 2 mM dissolved inorganic carbon (DIC), decreased sharply from pH 7.0 to 9.0, and was not substantially different from 0 above pH 9.0. These data indicate that P. purpurascens is inefficient in the use of external HCO₃ ⁻ as a carbon source in photosynthesis. Moreover, the photosynthetic rate as a function of external DIC and the highest pH (9.01 ± 0.07) that this species can achieve in a closed system were consistent with a low capacity to use HCO₃ ⁻, in comparison to many other species of seaweeds. The role of external carbonic anhydrase (CA; EC 4.2.1.1) on carbon uptake was investigated by measuring both the HCO₃ ⁻-dependent O₂ evolution and the CO₂ uptake, at pH 5.5 and 8.0, and the rate of pH change in the external medium, in the presence of selected inhibitors of extra- and intracellular CA. Photosynthetic DIC-dependent O₂ evolution was higher at pH 5.5 (where CO₂ is the predominant form of DIC) than at pH 8.0 (where the predominant chemical species is HCO₃ ⁻). Both intra- and extracellular CA activity was detected. Dextran-bound sulfonamide (DBS; a specific inhibitor of extracellular CA) reduced the photosynthetic O₂ evolution and CO₂ uptake at pH 8.0, but there was no effect at pH 5.5. The pH-change rate of the medium, under saturating irradiance, was reduced by DBS. Phyllariopsis purpurascens has a low efficiency in the use of HCO₃ ⁻ as carbon source in photosynthesis; nevertheless, the ion can be used after dehydration, in the external medium, catalyzed by extracellular CA. This mechanism could explain why the photosynthetic rate in situ was higher than that supported solely by the diffusion of CO₂ from seawater. Received: 6 March 1998 / Accepted: 22 June 1998     

Intracellular pH regulation and buffer capacity in CO2/HCO− 3-buffered media in cultured epithelial cells from rainbow trout gills

The influence of a CO₂/HCO⁻ ₃-buffered medium on intracellular pH regulation of gill pavement cells from freshwater rainbow trout was examined in monolayers grown in primary culture on glass coverslips; intracellular pH (pH_i) was monitored by continuous spectrofluorometric recording from cells loaded with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxy-fluoroscein. When cells in HEPES-buffered medium at normal pH=7.70 were transferred to normal CO₂/HCO⁻ ₃-buffered medium {P _CO2=3.71 mmHg, [HCO⁻ ₃]= 6.1 mmol l⁻¹, extracellular pH (pH_e)=7.70}, they exhibited a brief acidosis but subsequently regulated the same pHi (∼7.41) as in HEPES. Buffer capacity (β) increased by the expected amount (5.5–8.0 slykes) based on intracellular [HCO⁻ ₃], and was unaffected by most drugs and treatments. However, after transfer to high P _CO2=11.15 mmHg, [HCO⁻ ₃]= 18.2 mmol l⁻¹ at the same pH_e=7.70, the final regulated pHi was elevated (∼7.53). The rate of correction of alkalosis caused by washout of this high P _CO2, high-HCO⁻ ₃ medium was unaffected by removal of extracellular Cl⁻. Removal of extracellular Na⁺ lowered resting pH_i and greatly inhibited the rate of pH_i recovery from acidosis. Bafilomycin A₁ (3 μmol l⁻¹) had no effect on these responses. However amiloride (0.2 mmol l⁻¹) inhibited recovery from acidosis caused by washout of an ammonia prepulse, but did not affect resting pH_i, the latter differing from the response in HEPES where amiloride also lowered resting pH_i. Similarly 4-acetamido-4′- isothiocyanatostilbene-2,2′-disulfonic acid, sodium salt (0.1 mmol l⁻¹) did not affect resting pH_i but slowed the rate of recovery from acidosis, though to a lesser extent than amiloride. Removal of extracellular Cl⁻ also slowed the rate of recovery but greatly increased β by an unknown mechanism; when this was taken into account, H⁺ extrusion rate was unaffected. These results are consistent with the presence of Na⁺-(HCO⁻ ₃)_N co-transport and/or Na⁺-dependent HCO⁻ ₃/Cl⁻ exchange, in addition to Na⁺/H⁺ exchange, as mechanisms contributing to “housekeeping” pH_i regulation in gill cells in CO₂/HCO⁻ ₃ media, whereas only Na⁺/H⁺ exchange is seen in HEPES. Both Na⁺-independent Cl⁻/HCO⁻ ₃ exchange and V-type H⁺-ATPase mechanisms appear to be absent from these cells cultured in isotonic media. Accepted: 30 November 1999     

Role of Na+/ H+ exchange and HCO3 − transport in pHi recovery from intracellular acid load in cultured epithelial cells of sheep rumen

This study sought to investigate effects of short-chain fatty acids and CO₂ on intracellular pH (pH_i) and mechanisms that mediate pH_i recovery from intracellular acidification in cultured ruminal epithelial cells of sheep. pH_i was studied by spectrofluorometry using the pH-sensitive fluorescent indicator 2′,7′-bis (carboxyethyl)-5(6′)-carboxyfluorescein acetoxymethyl ester (BCECF/AM). The resting pH_i in N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES)-buffered solution was 7.37 ± 0.03. In HEPES-buffered solution, a NH₄ ⁺/NH₃-prepulse (20 mM) or addition of butyrate (20 mM) led to a rapid intracellular acidification (P < 0.05). Addition of 5-(N-ethyl-N-isopropyl)-amiloride (EIPA; 10 μM) or HOE-694 (200 μM) inhibited pH_i recovery from an NH₄ ⁺/NH₃-induced acid load by 58% and 70%, respectively. pH_i recovery from acidification by butyrate was reduced by 62% and 69% in the presence of EIPA (10 μM) and HOE-694 (200 μM), respectively. Changing from HEPES- (20 mM) to CO₂/HCO₃ ⁻-buffered (5%/20 mM) solution caused a rapid decrease of pH_i (P < 0.01), followed by an effective counter-regulation. 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS; 100 μM) blocked the pH_i recovery by 88%. The results indicate that intracellular acidification by butyrate and CO₂ is effectively counter-regulated by an Na⁺/H⁺ exchanger and by DIDS-sensitive, HCO₃ ⁻-dependent mechanism(s). Considering the large amount of intraruminal weak acids in vivo, both mechanisms are of major importance for maintaining the pH_i homeostasis of ruminal epithelial cells. Accepted: 8 March 2000     

Control of Cell pH in the T84 Colon Cell Line

Cell pH regulation was investigated in the T84 cell line derived from epithelial colon cancer. Cell pH was measured by ratiometric fluorescence microscopy using the fluorescent probe BCECF. Basal pH was 7.17 ± 0.023 (n= 48) in HEPES Ringer. After acidification by an ammonium pulse, cell pH recovered toward normal at a rate of 0.13 ± 0.011 pH units/min in the presence of Na⁺, but in the absence of this ion or after treatment with 0.1 mm hexamethylene amiloride (HMA) no significant recovery was observed, indicating absence of Na⁺ independent H⁺ transport mechanisms in HEPES Ringer. In CO₂/HCO⁻ ₃ Ringer, basal cell pH was 7.21 ± 0.020 (n= 35). Changing to HEPES Ringer, a marked alkalinization was observed due to loss of CO₂, followed by return to the initial pH at a rate of −0.14 ± 0.012 (n= 8) pH/min; this return was retarded or abolished in the absence of Cl⁻ or after addition of 0.2 mm DIDS, suggesting extrusion of bicarbonate by Cl⁻/HCO⁻ ₃ exchange. This exchange was not Na⁺ dependent. When Na⁺ was added to cells incubated in 0 Na⁺ Ringer while blocking Na⁺/H⁺ exchange by HMA, cell alkalinization by 0.19 ± 0.04 (n= 11) pH units was observed, suggesting the presence of Na⁺/HCO⁻ ₃ cotransport carrying HCO⁻ ₃ into these cells, which was abolished by DIDS. These experiments, thus, show that Na⁺/H⁺ and Cl⁻/HCO⁻ ₃ exchange and Na⁺/HCO⁻ ₃ cotransport participate in cell pH regulation in T84 cells. Received: 3 April 2000/Revised: 22 June 2000     

Na+-dependent HCO 3 − transport and Na+/H+ exchange regulate pH i in human ciliary muscle cells

Summary We investigated intracellular pH (pH _i ) regulation in cultured human ciliary muscle cells by means of the pH-sensitive absorbance of 5(and 6)-carboxy-4,5-dimethylfluorescein (CDMF). The steady-state pH _i was 7.09±0.04 (n = 12) in CO₂/ HCO ₃ ^– -buffered and 6.86±0.03 (n = 12) in HEPES-buffered solution. Removal of extracellular sodium for 6 min acidified the cells by 1.11±0.06 pH units (n = 12) in the presence of CO₂/ HCO ₃ ^– and by 0.91±0.05 pH units (n = 8) in its absence. Readdition of external sodium resulted in a rapid pH _i recovery, which was almost completely amiloride-sensitive in the absence of CO₂/ HCO ₃ ^– but only slightly influenced by amiloride in its presence. Application of DIDS under steady-state conditions significantly acidified the ciliary muscle cells by 0.25±0.02 (n = 4) in 6 min, while amiloride had no effect. The pH _i recovery after an intracellular acid load was completely dependent on extracellular sodium. In HEPES-buffered solution the pH _i recovery was almost completely mediated by Na⁺/H⁺ exchange, since it was blocked by amiloride (1 mmol/liter). In contrast, a marked amilorideinsensitive pH _i recovery was observed in CO₂/HCO ₃ ^– -buffered solution which was mediated by chloride-independent and chloride-dependent Na⁺ HCO ₃ ^– cotransport. This recovery, inhibited by DIDS (0.2 mmol/liter). was also observed if the cells were preincubated in chloride-free solution for 4 hr. Analysis of the sodium dependence of the pH _i recovery after NH₄Cl prepulse revealed V _max = 0.57 pH units/min, K _m= 39.7 mmol/liter extracellular sodium for the amiloride-sensitive component and V _max = 0.19 pH units/min, K _m= 14.3 mmol/liter extracellular sodium for the arniloride-insensitive component. We conclude that Na⁺/H⁺ exchange and chloride-independent and chloride-dependent Na⁺HCO ₃ ^– cotransport are involved in the pH _i regulation of cultured human ciliary muscle cells.The expert technical assistance of Astrid Krolik is gratefully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft grant DFG Wi 328/11.     

Photomineralization in a boreal hydroelectric reservoir: a comparison with natural aquatic ecosystems

In order to evaluate the role of photochemistry in the carbon dioxide (CO₂) generation from a 10-year-old boreal reservoir, the photomineralization of dissolved organic matter (DOM) was assessed and compared to a boreal river as well as to boreal and temperate lakes during July and August, 2003. Sterile water samples were irradiated by sunlight over the whole photoperiod and subsequently analyzed for CO₂. Mean energy-normalized apparent photochemical yield of CO₂ (an index of DOM photoreactivity normalized for the energy absorbed by samples) was significantly higher in the reservoir (27.7 ± 13.0 mg CO₂·m⁻³·kJ⁻¹) and the boreal river (35.8 ± 2.3 mg CO₂·m⁻³·kJ⁻¹) than in the boreal lakes (15.5 ± 5.1 mg CO₂·m⁻³·kJ⁻¹). The DOM photoreactivity of the temperate lakes (20.9 ± 8.1 mg CO₂·m⁻³·kJ⁻¹) was not statistically different from any type of boreal water bodies. There was no significant difference in either the integrated photoproduction of CO₂ (273–433 mg CO₂·m⁻²·d⁻¹) or the potential photochemical contribution to CO₂ diffusive fluxes (56–92%) among these water bodies. DOM photoreactivity was significantly affected by the cumulative hydrological residence time (CHRT) when considering the whole data set. However, when considering only the boreal water bodies, iron (Fe) and manganese (Mn) also intervened. The fact that DOM photoreactivity was related to CHRT as well as to Fe and Mn concentrations, which are respectively permanent and long-lasting features of the reservoir, suggests that the photoproduction of CO₂ is not likely to decrease over time. This process may therefore play a substantial role in the long-term CO₂ emissions from boreal reservoirs during the summer, its potential contribution to CO₂ diffusive fluxes being estimated at 56 ± 29 %.     

Bicarbonate,the most important factor inducing iron chlorosis in vine grapes on calcareous soil

Summary In pot experiments grape vine was grown on a calcareous and on a non calcareous soil with a low and with a high water saturation. During the growing period soil solution samples were collected and analyzed for their pH and for HCO ₃ ⁻ , phosphate, Fe, and Ca. High water saturation resulted in a pH increase and in an increase of the HCO ₃ ⁻ concentration in both soils. The level in pH and HCO ₃ ⁻ , however, was much higher in the calcareous soil than in the non calcareous soil. The Fe concentration varied much throughout the experimental period, but there was no major differences between soils and water saturation treatments. The Ca concentration of the soil solution increased with time in the calcareous soil; for the non calcareous soil rather the reverse was true. The phosphate level in the soil solution of the non calcareous soil was about 10 times higher than in the calcareous soil. After 3 weeks growth all plants of the calcareous soil with the high water saturation showed first symptoms of Fe deficiency. These became more intense from day to day. Plants of the other treatments did not show any chlorotic symptoms. In the treatment with the chlorotic plants the HCO ₃ ⁻ concentration of the soil solution was the highest, the phosphate concentration the lowest from all treatments. It is therefore concluded that HCO ₃ ⁻ and not phosphate is the primary cause for lime induced Fe chlorosis. Despite the low phosphate concentration in the soil solution, the P concentration in the chlorotic leaves was more than twice as high as the P concentration in green leaves grown on the same soil. It is thus assumed that the high P content frequently found in chlorotic leaves is the result and not the cause for Fe chlorosis.     

Control of nutrient solutions for studies at high pH

Little is known about factors effecting plant growth at high pH, with research often limited by the inability to separate nutritional deficiencies and HCO ₃ ⁻ toxicity from the direct limitations imposed under high pH conditions. Various methods of controlling dilute nutrient solutions for studies at high pH were investigated. For short-term studies, it was found that a solution without Cu, Fe, Mn and Zn and aerated with CO₂ depleted air, greatly reduced nutrient precipitation at high pH, thus eliminating nutritional differences between treatments. Manual pH adjustment and the use of ion exchange resins as pH buffers were unsuitable methods of pH control. However, pH control by automated titration had little effect on solution composition while maintaining constant pH. The system described is suitable for studies in which the pH of the bulk nutrient solution must be maintained. The system was used to examine OH⁻ toxicity in mungbeans (Vigna radiata (L.) Wilczek cv. Emerald), with root length reduced at a bulk solution pH of 8.5 and greater.     

N2O, CH4 and CO2 emissions from seasonal tropical rainforests and a rubber plantation in Southwest China

The main focus of this study was to evaluate the effects of soil moisture and temperature on temporal variation of N₂O, CO₂ and CH₄ soil-atmosphere exchange at a primary seasonal tropical rainforest (PF) site in Southwest China and to compare these fluxes with fluxes from a secondary forest (SF) and a rubber plantation (RP) site. Agroforestry systems, such as rubber plantations, are increasingly replacing primary and secondary forest systems in tropical Southwest China and thus effect the N₂O emission in these regions on a landscape level. The mean N₂O emission at site PF was 6.0 ± 0.1 SE μg N m⁻² h⁻¹. Fluxes of N₂O increased from <5 μg N m⁻² h⁻¹ during dry season conditions to up to 24.5 μg N m⁻² h⁻¹ with re-wetting of the soil by the onset of first rainfall events. Comparable fluxes of N₂O were measured in the SF and RP sites, where mean N₂O emissions were 7.3 ± 0.7 SE μg N m⁻² h⁻¹ and 4.1 ± 0.5 SE μg N m⁻² h⁻¹, respectively. The dependency of N₂O fluxes on soil moisture levels was demonstrated in a watering experiment, however, artificial rainfall only influenced the timing of N₂O emission peaks, not the total amount of N₂O emitted. For all sites, significant positive correlations existed between N₂O emissions and both soil moisture and soil temperature. Mean CH₄ uptake rates were highest at the PF site (−29.5 ± 0.3 SE μg C m⁻² h⁻¹), slightly lower at the SF site (−25.6 ± 1.3 SE μg C m⁻² h⁻¹) and lowest for the RP site (−5.7 ± 0.5 SE μg C m⁻² h⁻¹). At all sites, CH₄ uptake rates were negatively correlated with soil moisture, which was also reflected in the lower uptake rates measured in the watering experiment. In contrast to N₂O emissions, CH₄ uptake did not significantly correlate with soil temperature at the SF and RP sites, and only weakly correlated at the PF site. Over the 2 month measurement period, CO₂ emissions at the PF site increased significantly from 50 mg C m⁻² h⁻¹ up to 100 mg C m⁻² h⁻¹ (mean value 68.8 ± 0.8 SE mg C m⁻² h⁻¹), whereas CO₂ emissions at the SF and RP site where quite stable and varied only slightly around mean values of 38.0 ± 1.8 SE mg C m⁻² h⁻¹ (SF) and 34.9 ± 1.1 SE mg C m⁻² h⁻¹ (RP). A dependency of soil CO₂ emissions on changes in soil water content could be demonstrated for all sites, thus, the watering experiment revealed significantly higher CO₂ emissions as compared to control chambers. Correlation of CO₂ emissions with soil temperature was significant at the PF site, but weak at the SF and not evident at the RP site. Even though we demonstrated that N and C trace gas fluxes significantly varied on subdaily and daily scales, weekly measurements would be sufficient if only the sink/ source strength of non-managed tropical forest sites needs to be identified.     

Growth and net photosynthetic rates of <Emphasis Type="Italic">Hosta</Emphasis> ‘blue vision’ during acclimatization in bright,natural light with CO<Subscript>2</Subscript> enrichment

Summary Hosta ‘Blue Vision’, a shade-adapted perennial, was successfully acclimatized in high, natural light conditions in the research Acclimatron^TM at Clemson University, Clemson, SC during the summer of 2000. The supplemental CO₂ levels achieved during acclimatization were 710±113, 2396±121, and 5641±119 μmol mol⁻¹, approximately 2×, 6×, and 15× ambient CO₂. Plants were maintained in H₂O-saturated atmospheres and protected from temperature increases associated with high light intensity. In the 5 wk following ex vitro transfer, plantlet roots grew at the 2× CO₂ level, but shoot biomass was unaffected. Results for the 6× and 15× CO₂ levels were comparable and provided the best plantlet growth. The “doubling time’ that is characteristic of exponential growth was 10.8 and 9.8 d for root and shoot dry weights, respectively. There was no indication of light saturation of net photosynthetic rate (NPR) over the photosynthetic photon flux density (PPFD) range of 100–1200 μmolm⁻²s⁻¹ experienced during this study. An interaction between CO₂ and light intensity levels was detected for NPR of Hosta ‘Blue Vision’ with CO₂ saturation occurring at approximately 2800 μmol mol⁻¹. regardless of light level. Furthermore, at the optimal CO₂ level, NPR increased quadratically as light intensity increased, and NPR was greatest at the maximum light intensity (PPFD: 1200 μmol m⁻²s⁻¹).     

Carbonate precipitates and bicarbonate secretion in the intestine of sea bass, <Emphasis Type="Italic">Dicentrarchus labrax</Emphasis>

The aim of this paper was to study the chemical composition of the precipitates found in the intestine of Dicentrarchus labrax and the source of HCO₃ ⁻ secreted into the intestinal lumen. The chemical analysis was performed by employing the potentiometric double titration method and by means of an electron microscope coupled with a spectrometer and X-ray powder diffraction. The results obtained suggest the presence of very insoluble intestinal precipitates, presumably formed by a mixture of CaCO₃ and MgCO₃, with a higher quantity of the former with respect to the latter. HCO₃ ⁻ secretion rate was investigated with the aid of the pH stat method in isolated tissues mounted in Ussing chamber, where the transepithelial electrical parameters were also measured. When the serosal surface of the intestinal mucosa was bathed in HCO₃ ⁻-Ringer bubbled with 1% CO₂ in O₂ while the serosal surface was bathed in HCO₃ ⁻ free Ringer solution bubbled with pure O₂, bicarbonate secretion proceeded at an almost stable rate of 0.9 ± 0.05 μeq cm⁻² h⁻¹ for about 3 h while I _sc maintained a constant value of 38 ± 1.5 μA cm⁻². The carbonic anhydrase inhibitor ethoxyzolamide elicited a progressive reduction of HCO₃ ⁻ secretion that was about 75% of the initial value after 80 min. When serosal HCO₃ ⁻–CO₂ saline was substituted with Hepes–O₂ saline base secretion progressively declined reaching a value of about 20% of the initial value. It was also strongly inhibited when Na⁺ was substituted with the impermeant cation choline and when either DIDS or ouabain were added to the basolateral side. These results suggest that most of the bicarbonate secreted is of extracellular source and is probably transported across the basolateral membrane by both Na⁺ independent mechanism and Na⁺ dependent transporter, presumably a NaHCO₃ cotransport.     

Response of soil respiration to simulated N deposition in a disturbed and a rehabilitated tropical forest in southern China

Responses of soil respiration (CO₂ emission) to simulated N deposition were studied in a disturbed (reforested forest with previous understory and litter harvesting) and a rehabilitated (reforested forest with no understory and litter harvesting) tropical forest in southern China from October 2005 to September 2006. The objectives of the study were to test the following hypotheses: (1) soil respiration is higher in rehabilitated forest than in disturbed forest; (2) soil respiration in both rehabilitated and disturbed tropical forests is stimulated by N additions; and (3) soil respiration is more sensitive to N addition in disturbed forest than in rehabilitated forest due to relatively low soil nutrient status in the former, resulting from different previous human disturbance. Static chamber and gas chromatography techniques were employed to quantify the soil respiration, following different N treatments (Control, no N addition; Low-N, 5 g N m⁻² year⁻¹; Medium-N, 10 g N m⁻² year⁻¹), which had been applied continuously for 26 months before the respiration measurement. Results showed that soil respiration exhibited a strong seasonal pattern, with the highest rates observed in the hot and wet growing season (April–September) and the lowest rates in winter (December–February) in both rehabilitated and disturbed forests. Soil respiration rates exhibited significant positive exponential relationship with soil temperature and significant positive linear relationship with soil moisture. Soil respiration was also significantly higher in the rehabilitated forest than in the disturbed forest. Annual mean soil respiration rate in the rehabilitated forest was 20% lower in low-N plots (71 ± 4 mg CO₂-C m⁻² h⁻¹) and 10% lower in medium-N plots (80 ± 4 mg CO₂-C m⁻² h⁻¹) than in the control plots (89 ± 5 mg CO₂-C m⁻² h⁻¹), and the differences between the control and low-N or medium-N treatments were statistically significant. In disturbed forest, annual mean soil respiration rate was 5% lower in low-N plots (63 ± 3 mg CO₂-C m⁻² h⁻¹) and 8% lower in medium-N plots (61 ± 3 mg CO₂-C m⁻² h⁻¹) than in the control plots (66 ± 4 mg CO₂-C m⁻² h⁻¹), but the differences among treatments were not significant. The depressed effects of experimental N deposition occurred mostly in the hot and wet growing season. Our results suggest that response of soil respiration to elevated N deposition in the reforested tropical forests may vary depending on the status of human disturbance. Responsible Editor: Hans Lambers.     

Soil surface CO2 flux in a Minnesota peatland

Soil surface CO₂ flux was measured in hollow and hummock microhabitats in a peatland in north central Minnesota from June to October in 1991. We used a closed infrared gas exchange system to measure soil CO₂ flux. The rates of CO₂ evolution from hummocks (9.8 ± 3.5 g m⁻² d⁻¹, [mean ± SE]) were consistently higher than those from hollows (5.4 ± 2.9 g m⁻² d⁻¹) (the hummock values included the contribution of moss dark respiration, which may account for 10–20% of the total measured flux). The soil CO₂ flux was strongly temperature-dependent (Q₁₀ ≈ 3.7) and appeared to be linearly related to changes in water table depth. An empirical multiplicative model, using peat temperature and water table depth as independent variables, explained about 81% of the variance in the CO₂ flux data. Using the empirical model with measurements of peat temperature and estimates of hollow/hummock microtopographic distribution (relative to water table elevation), daily rates of “site-averaged” CO₂ evolution were calculated. For the six-month period (May–October), the total soil CO₂ released from this ecosystem was estimated to be about 1340 g CO₂ m⁻². Published as Paper No. 9950, Journal Series, Nebraska Agricultural Research Division, University of Nebraska, Lincoln, NE, USA.     

Stimulated N2O flux from intact grassland monoliths after two growing seasons under elevated atmospheric CO2

Long-term exposure of native vegetation to elevated atmospheric CO₂ concentrations is expected to increase C inputs to the soil and, in ecosystems with seasonally dry periods, to increase soil moisture. We tested the hypothesis that these indirect effects of elevated CO₂ (600 μl l⁻¹ vs 350 μl l⁻¹) would improve conditions for microbial activity and stimulate emissions of nitrous oxide (N₂O), a very potent and long-lived greenhouse gas. After two growing seasons, the mean N₂O efflux from monoliths of calcareous grassland maintained at elevated CO₂ was twice as high as that measured from monoliths maintained at current ambient CO₂ (70 ± 9 vs 37 ± 4 μg N₂O m⁻² h⁻¹ in October, 27 ± 5 vs 13 ± 3 μg N₂O m⁻² h⁻¹ in November after aboveground harvest). The higher N₂O emission rates at elevated CO₂ were associated with increases in soil moisture, soil heterotrophic respiration, and plant biomass production, but appear to be mainly attributable to higher soil moisture. Our results suggest that rising atmospheric CO₂ may contribute more to the total greenhouse effect than is currently estimated because of its plant-mediated effects on soil processes which may ultimately lead to increased N₂O emissions from native grasslands. Received: 11 September 1997 / Accepted: 20 March 1998     

Synergistic control of CO2 emissions by fish and nutrients in a humic tropical lake

Using experimental mesocosms, we tested the strength of bottom–up controls by nutrients and top–down controls by an omnivorous fish (Hyphessobrycon bifasciatus; family Characidae), and the interaction between them on the CO₂ partial pressure (pCO₂) in the surface waters of a tropical humic lake (Lake Cabiúnas, Brazil). The experiment included the addition of nutrients and fish to the mesocosms in a factorial design. Overall, persistent CO₂ emissions to the atmosphere, supported by an intense net heterotrophy, were observed in all treatments and replicates over the 6-week study period. The CO₂ efflux (average ± standard error) integrated over the experiment was similar among the control mesocosms and those receiving only fish or only nutrients (309 ± 2, 303 ± 16, and 297 ± 17 mmol CO₂ m⁻² day⁻¹, respectively). However, the addition of nutrients in the presence of fish resulted in a high algal biomass and daytime net autotrophy, reducing the CO₂ emissions by 35% (by 193 ± 7 mmol CO₂ m⁻² day⁻¹). These results indicate that high CO₂ emissions persist following the eutrophication of humic waters, but that the magnitude of these emissions might depend on the structure of the food web. In conclusion, fish and nutrients may act in a synergistic manner to modulate persistent CO₂ emissions from tropical humic lakes.