Carbon dioxide excretion and carbonic anhydrase function in the Red Rock CrabCancer productus

Summary The function of carbonic anhydrase (CA) in the Red Rock Crab,Cancer productus Randall, was investigated. CA activity was found to varying degrees in the gills and many other tissues but not in hemolymph. Crabs injected with acetazolamide, a specific CA inhibitor, demonstrated a significant hemolymph acidosis. Hemolymph CO₂ tension ( ) and CO₂ content ( ) also increased and remained significantly elevated for 96 h following treatment. No significant changes could be detected in either hemolymph oxygenation or ionic status (except for HCO ₃ ^– ) as a result of acetazolamide treatment. Crabs treated with acetazolamide, and also exposed to air, exhibited a more pronounced hemolymph acidosis with significantly increased respiratory ( ) and metabolic (lactate) components compared with the control group. Upon reimmersion acetazolamide treated crabs showed a slower recovery of hemolymph pH compared with the control group and no significant removal of the total CO₂ load induced by air exposure. No significant differences between experimental and control groups during air exposure and recovery could be detected in hemolymph oxygenation, ionic status, NH₃+NH ₄ ⁺ levels or respiratory and cardiac pumping frequency and so the effects of acetazolamide treatment were apparently limited to CO₂ removal across the gills. These results indicate that branchial CA facilitates the removal of CO₂ from the hemolymph of SW adaptedC. productus largely by catalyzing the dehydration of hemolymph HCO ₃ ^– to molecular CO₂ at the gill. It is also recognized that gill CA may also serve to hydrate molecular CO₂ to H⁺ and HCO_3/– for use as counterions for ionic uptake mechanisms. Crab gill CA thus appears to play an important role in CO₂ excretion as well as hemolymph ionic regulation.     

Aerial and Aquatic Respiration in the River Crab Potamonautes Warreni Calman with Notes on Gill Structure

Summary

The oxygen consumption rate (?O₂) for Potamonauteus warreni Calman (= Potamon warreni (Calman) kept in 25 °C water was 34,4 μmol 1^?1 O₂ kg^?1 and after 72 hours in 98% R.H. air the rate was 31,9 μmol 1^?1 O₂ kg^?1 min^?1. The ?O₂ values for each of the two groups are not significantly different (P > 0,05). The partial oxygen tension of pre-branchial (v = venous) haemolymph (P_vCO₂) is 15,3 mm Hg in water and 13,0 mm Hg in air); partial carbon dioxide tension of pre-branchial (v) haemolymph (P_vCO₂) is 13,2 mm Hg in water and 13,0 mm Hg in air); the total carbon dioxide concentration in pre-branchial (v) haemolymph (C_vCO₂) tot. is 12,3 mmol 1^?1 in air and 13,9 mmol 1^?1 in water) are not significantly different for the two groups (P > 0,05). The haemolymph pH and the lactate concentration for crabs in water was found to be 7,51 and 0,38 mmol 1^?1 respectively. No significant differences were found in pre-branchial haemolymph oxygen tension, carbon dioxide tension, total carbon dioxide content, haemolymph pH, lactate level, chloride concentration, P₅₀ and haemocyanin-oxygen cooperativity in control crabs kept in water, and experimental crabs held in air for 72 hours. The chloride concentration, (327,0 mmol 1^?1) for crabs kept in water does not differ from that of crabs held in air for 72 hours but is at least 15% higher than the sodium concentration (255 mmol 1^?1) for crabs kept in water. The gill surface area is 520 mm² g^?1 wet body mass; on average 9,2 gill platelets (lamellae) can be found on a gill length of one millimetre. Each lamella is spaced 60–70 μm apart, each with a thickness of 30–40 μm. It is concluded that P. warreni may be described as a truly amphibious fresh-water crab.     

Hemolymph oxygen content,oxyhemocyanin, protein levels and ammonia excretion in the shrimp Penaeus monodon exposed to ambient nitrite

Subadult Penaeus monodon (21.03±3.19 g) were exposed individually in sea water (30 mg·ml^-1) to 0.02 (control), 1.04, 5.02, 10.11 and 20.06 mg·l^-1 nitrite-N for 24h. Hemolymph pH, partial pressures of oxygen and carbon dioxide, bicarbonate concentration, oxyhemocyanin and protein levels, and whole animal ammonia-N excretion and nitrite-N uptake were determined. Ammonia-N excretion and hemolymph oxygen partial pressure increased, whereas hemolymph pH, HCO ₃ ^- , oxyhemocyanin, protein and the ratio of oxyhemocyanin/protein levels decreased with increasing ambient nitrite-N. It is suggested that accumulated nitrite of P. monodon following exposure to ambient nitrite causes reduction of oxyhemocyanin, protein and the ratio of oxyhemocyanin/protein in the hemolymph, and affects nitrogen metabolism and acid-base balance at low hemolymph pH.Abbreviations bw body weight - EC₅₀ concentration reducing growth rate by 50% that of controls - LC₅₀ median lethal concentration - nitrite-N nitrite concentration measured as nitrogen - PO₂ partial pressure of O₂ in hemolymph - PCO₂ partial pressure of CO₂ in hemolymph - sw sea water - ww wet weight     

Respiratory,cardiovascular, and hemolymph acid‐base changes in the amphibious crab,Cardisoma guanhumi,during immersion and emersion

The cardiorespiratory and hemolymph acid base status of bimodal breathing crabs, Cardisoma guanhumi, was monitored during the transition from breathing air to breathing water. Upon immersion, oxygen uptake (MO₂) decreased by half. Ventilatory frequency (fsc) increased more than 5 fold, causing a decrease in hemolymph carbon dioxide partial pressure (PCO₂). This was nearly fully compensated for by a gradual decrease in hemolymph bicarbonate concentration ([HCO₃ ^‐]) over 96 hours post‐immersion. After one to two weeks of immersion, when crabs were removed from the water, oxygen uptake initially increased, but eventually returned to the initial immersed value. Heart rate was unchanged but fsc slowed dramatically. The decreased ventilation resulted in a buildup of hemolymph PCO₂, causing a respiratory acidosis that was slowly compensated for by increased hemolymph [HCO₃ ^‐]. C. guanhumi appears to be a truly amphibious crab with respiratory and acid‐base adaptations found in both fully aquatic and fully terrestrial species.     

Ammonia exposure of Chasmagnathus granulata (Crustacea, Decapoda) Dana, 1851: accumulation in haemolymph and effects on osmoregulation

In order to study lethal and sublethal effects of ammonia to the estuarine crab Chasmagnathus granulata in the presence of an additional stress factor such as salinity, we determined the LC₅₀ (96 h) of ammonia at 20‰ and in response to osmotic stress (5–40‰) and evaluated ammonia accumulation in the haemolymph of C. granulata and ammonia effects on osmo- and ion-regulation of this species through determinations of the haemolymph Na⁺, Ca²⁺, Cl⁻ and osmotic concentration. The LC₅₀ values (96 h) of total ammonia (NH₃+NH₄⁺) were 10.10, 17.85 and 14.0 mM for crabs maintained at 5, 20 or 40‰ salinity, respectively, suggesting that this crab is fairly resistant to ammonia. The haemolymph ammonia concentration augmented with ambient ammonia during a 6-h exposure to sublethal ammonia concentrations which were not enough to reach equilibrium between external and haemolymph ammonia. At 20‰ salinity, following a 96-h exposure to sublethal concentrations, a significant decrease (P<0.05) of haemolymphatic chloride concentration was registered at 3.3 and 5.5 mM of total ammonia. At 40‰ salinity, a significant increase (P<0.05) of the haemolymph osmotic pressure was apparent at 5.5 mM total ammonia. We postulate that C. granulata gives priority to NH₃ formation as a mechanism to eliminate it by simple diffusion. The differential Na⁺ and Cl⁻ regulation of crabs maintained at 20‰ salinity could modify the strong ion difference, augmenting pH, which in turn should lead the NH₄⁺/NH₃ equilibrium towards NH₃.     

Effects of azadirachtin on six inorganic cation distributions in Ostrinia furnacalis (G.)

Azadirachtin (Az), as a botanical insecticide, is relatively safe and biodegradable. It affects a wide vaariety of biological processes, including the reduction of feeding, suspension of molting, death of larvae and pupae, and sterility of emerged adults in a dose-dependent manner. However, the mode of action of this toxin remains obscure. By using ion chromatography, we analyzed changes in six inorganic cation (Li⁺, Na⁺, NH₄ ⁺, K⁺, Mg²⁺, and Ca²⁺) distributions of the whole body and hemolymph in Ostrinia furnacalis (G.) after exposure to sublethal doses of Az. The results showed that Az dramatically interfered with Na⁺, NH₄ ⁺, K⁺, Mg²⁺, and Ca²⁺ distributions in hemolymph of O. furnacalis (G.) and concentrations of these five cations dramatically increased. However, in the whole body, the levels of K⁺, Mg²⁺, and Ca²⁺ significantly, decreased after exposure to Az, except that Na⁺ and NH₄ ⁺ remained constant. Li⁺ was undetected in both the control and treated groups in the whole body and hemolymph. It is suggested that Az exerts its insecticidal effects on O. furnacalis (G.) by interfering with the inorganic cation distributions related to ion channels.     

土壤氮水交互对马尾松和杉木COS和CO2通量的影响

羰基硫(COS)和CO₂化学结构相似,且植物对COS和CO₂具有共吸收特性,因此可利用COS作为示踪物来估算生态系统总初级生产力,而不同植物吸收COS和CO₂对环境因子变化的响应差异较大。以南亚热带典型树种马尾松(Pinus massoniana)和杉木(Cunninghamia lanceolata)为研究对象,设置2个氮水平及3个土壤水分梯度处理。采取顶空套袋法采集气体样品,用预浓缩—气质联用仪分析样品COS浓度,同时测量植物光合参数。结果表明:马尾松和杉木吸收COS,吸收速率均值分别为39.58—127.27 pmol m^-2 s^-1和0.81—66.92 pmol m^-2 s^-1。整体而言,施氮可促进植物吸收COS,但除施氮对马尾松COS通量有显著影响外(P<0.05),施氮、土壤水分和两者交互作用对马尾松和杉木的COS和CO₂通量及其比值均无显著性影响。施氮情况下,高土壤水分处理促进马尾松COS吸...     

Effect of starvation and refeeding on amino acid metabolism in muscle of crab Neohelice granulata previously fed protein- or carbohydrate-rich diets

The present study assesses the effects of starvation and refeeding on 1-[¹⁴C]-methyl aminoisobutyric acid (¹⁴C-MeAIB) uptake, ¹⁴C-total lipids, ¹⁴CO₂ production from ¹⁴C-glycine, ¹⁴C-protein synthesis from ¹⁴C-leucine and Na⁺–K⁺-ATPase activity in jaw muscle of Neohelice granulata previously maintained on a carbohydrate-rich (HC) or high-protein (HP) diet. In N. granulata the metabolic adjustments during starvation and refeeding use different pathways according to the composition of the diet previously offered to the crabs. During starvation, ¹⁴CO₂ production from ¹⁴C-glycine, and ¹⁴C-protein synthesis from ¹⁴C-leucine were reduced in HC-fed crabs. In crabs maintained on the HP or HC diet, ¹⁴C-total lipid synthesis increased after 15 days of starvation. In crabs fed HP diet, ¹⁴C-MeAIB uptake and Na⁺–K⁺-ATPase activity decreased in refeeding state. In crabs refeeding HC diet, ¹⁴C-MeAIB uptake and ¹⁴CO₂ production decreased during the refeeding. In contrast, the ¹⁴C-protein synthesis increased after 120 h of refeeding. In both dietary groups, ¹⁴C-total lipid synthesis increased during refeeding. Changes in the carbon amino acid flux between different metabolic pathways in muscle are among the strategies used by this crab to face starvation and refeeding. Protein or carbohydrate levels in the diet administered to this crab modulate the carbon flux between the different metabolic pathways.     

Salinity-mediated carbonic anhydrase induction in the gills of the euryhaline green crab, Carcinus maenas

The euryhaline green crab, Carcinus maenas, is a relatively strong osmotic and ionic regulator, being able to maintain its hemolymph osmolality as much as 300 mOsm higher than that in the medium when the crab is acclimated to low salinity. It makes the transition from osmoconformity to osmoregulation at a critical salinity of 26 ppt, and new acclimated concentrations of hemolymph osmotic and ionic constituents are reached within 12 h after transfer to low salinity. One of the central features of this transition is an 8-fold induction of the enzyme carbonic anhydrase (CA) in the gills. This induction occurs primarily in the cytoplasmic pool of CA in the posterior, ion-transporting gills, although the membrane-associated fraction of CA also shows some induction in response to low salinity. Inhibition of branchial CA activity with acetazolamide (Az) has no effect in crabs acclimated to 32 ppt but causes a depression in hemolymph osmotic and ionic concentrations in crabs acclimated to 10 ppt. The salinity-sensitive nature of the cytoplasmic CA pool and the sensitivity of hemolymph osmotic/ionic regulation to Az confirm the enzyme's role in ion transport and regulation in this species. CA induction is a result of gene activation, as evidenced by an increase in CA mRNA at 24 h after transfer to low salinity and an increase in protein-specific CA activity immediately following at 48 h post-transfer. CA gene expression appears to be under inhibitory control by an as-yet unidentified repressor substance found in the major endocrine complex of the crab, the eyestalk.     

Net ecosystem production of a Douglas-fir stand for 3 years following clearcut harvesting

To investigate the variations in annual and seasonal net ecosystem production (F_NEP) during the development of a young forest, 3 years of continuous eddy covariance measurements of carbon dioxide (CO₂) fluxes were collected following clearcut harvesting and replanting of a coastal Douglas‐fir stand on the east coast of Vancouver Island, BC, Canada. The impact of changing weather and stand structure on F_NEP was examined by developing relationships between F_NEP and variables such as light, temperature, soil moisture, and leaf area index (LAI). In all 3 years, the stand was a large source of CO₂ (620, 520, and 600 g C m^?2 yr^?1 in the first, second, and third years, respectively). During this period, the growth of pioneer and understory species resulted in an increase in maximum growing season LAI from 0.2 in the year the seedlings were planted to 2.5 in the third year. The associated increase in annual gross ecosystem production (P=F_NEP?R_e, where R_e is ecosystem respiration) from 220 g C m^?2 yr^?1 in the first year to 640 g C m^?2 yr^?1 in the third year was exceeded by an increase in annual R_e from 840 to 1240 g C m^?2 yr^?1. Seasonal and interannual variations in daytime F_NEP and P were well described by variations in photosynthetically active radiation, temperature, and changes in LAI. Night‐time measurements of R_e exponentially increased with 2 cm soil temperature with an average Q₁₀ of 2 (relative increase in R_e for a 10°C increase in temperature) and R₁₀ (R_e at 10°C) that increased from 2.1 in the first year to 2.5 in the second year to 3.2 μmol m^?2 s^?1 in the third year. Although the re‐establishment of vegetation in this stand had a major impact on both P and R_e, interannual variations in weather also affected annual F_NEP. Drought, in the summer of the third year, resulted in early senescence and reduced both P and R_e. This resulted in more C being lost from the stand in the third year after harvesting than in the second year.     

Production,purification, and characterization of a fusion protein of carbonic anhydrase from Neisseria gonorrhoeae and cellulose binding domain from Clostridium thermocellum

Carbon dioxide capture technologies have the potential to become an important climate change mitigation option through sequestration of gaseous CO₂. A new concept for CO₂ capture involves use of immobilized carbonic anhydrase (CA) that catalyzes the reversible hydration of CO₂ to HCO₃^? and H⁺. Cost‐efficient production of the enzyme and an inexpensive immobilization system are critical for development of economically feasible CA‐based CO₂ capture processes. An artificial, bifunctional enzyme containing CA from Neisseria gonorrhoeae and a cellulose binding domain (CBD) from Clostridium thermocellum was constructed with a His₆ tag. The chimeric enzyme exhibited both CA activity and CBD binding affinity. This fusion enzyme is of particular interest due to its binding affinity for cellulose and retained CA activity, which could serve as the basis for improved technology to capture CO₂ from flue gasses. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Sap flow in Scots pines growing under conditions of year-round carbon dioxide enrichment and temperature elevation

Starting in rrrr, individual trees of Scots pine (Pinus sylvestris L.) aged 30 years were grown in closed-top chambers and exposed to normal ambient conditions (CON), elevated CO₂ (Elev. C), elevated temperature (Elev. T) and a combination of elevated CO₂ and temperature (Elev. C + T). Using the constant-power heat balance method, sap flow was monitored simultaneously in a total of 16 trees, four for each treatment, over a 32 d period (after the completion of needle expansion and branch elongation in 1997). An overall variation in diurnal sap flow totals (F_t) was evident during the period of measurement (days 167–198, 1997) regardless of the treatments, with a range from 0·15 to 2·82 kg tree^–1 d^–1. Elev. C reduced F_t by 4·1–13·7% compared with CON on most days (P varies from 0·042 to 0·108), but slightly increased it on some days (P≥ 0·131), depending on the weather conditions. Although the decrease in F_t caused by Elev. C was statistically significant on only a few days (P≤ 0·042), the cumulative F_t for the 32 d decreased by 14·4% (P = 0·047), indicating that Elev. C may have an important influence on seasonal water use of the Scots pine. Analysis of the diurnal courses of sap flow combined with corresponding weather factors indicated that the CO₂-induced decrease in F_t could be largely attributed to an increase in stomatal sensitivity to vapour pressure deficit (VPD), whereas the CO₂-induced increase in F_t related to an increase in stomatal sensitivity to low light levels. Elev. T increased F_t by 11·2–35·6% throughout the measuring period and the cumulative F_t for the 32 d by 32·5% (P = 0·019), which could be largely attributed to the temperature-induced increase in current-year needle area and decrease in stomatal sensitivity to high levels of VPD. There were no significant interactive effects of CO₂ and temperature on sap flow, so that Elev. C + T had approximately the same F_t as Elev. T and similar diurnal patterns of sap flow, suggesting that the temperature factor played a dominant role in the case of Elev. C + T.     

A chlorophyll fluorescence analysis of photosynthetic efficiency, quantum yield and photon energy dissipation in PSII antennae of Lactuca sativa L. leaves exposed to cinnamic acid

This study investigated the effects of cinnamic acid (CA) on growth, biochemical and physiological responses of Lactuca sativa L. CA (0.1, 0.5, 1.0 and 1.5 mM) treatments decreased plant height, root length, leaf and root fresh weight, but it did not affect the leaf water status. CA treatment (1.5 mM) significantly reduced F_v, F_m, photochemical efficiency of PSII (F_v/F_m) and quantum yield of PSII (ΦPSII) photochemistry in L. sativa. The photochemical fluorescence quenching (qP) and non-photochemical quenching (NPQ) were reduced after treatment with 1.5 mM CA. Fraction of photon energy absorbed by PS II antennae trapped by “open” PS II reaction centers (P) was reduced by CA (1.5 mM) while, portion of absorbed photon energy thermally dissipated (D) and photon energy absorbed by PSII antennae and trapped by “closed” PSII reaction centers (E) was increased. Carbon isotope composition ratios (δ¹³C) was less negative (−27.10) in CA (1.5 mM) treated plants as compared to control (−27.61). Carbon isotope discrimination (Δ¹³C) and ratio of intercellular CO₂ concentration (ci/ca) from leaf to air were also less in CA treated plants. CA (1.5 mM) also decreased the leaf protein contents of L. sativa as compared to control.     

Effect of hyper or hypo-osmotic conditions on neutral amino acid uptake and oxidation in tissues of the crab Chasmagnathus granulata

We investigated the transport of ¹⁴C-methylaminoisobutyric acid (¹⁴C-MeAIB) and ¹⁴C-alanine oxidation in hepatopancreas and jaw muscle of Chasmagnathus granulata submitted to 24, 72, and 144 h of hypo- or hyperosmotic stress. While ¹⁴C-MeAIB uptake increased in jaw muscle and hepatopancreas from crabs submitted to hyperosmotic stress, it did not change in tissues from animals submitted to hypo-osmotic stress. Incubation of jaw muscle and hepatopancreas from control groups with 1 mM ouabain did not decrease ¹⁴C-MeAIB uptake. However, ouabain prevented ¹⁴C-MeAIB uptake in hepatopancreas at 24 h of hyperosmotic stress. In contrast, in jaw muscle from crabs submitted to the same conditions, ¹⁴C-MeAIB uptake was not prevented by ouabain in the incubation medium. Jaw muscle from the control group produced four times more ¹⁴CO₂ from ¹⁴C-alanine than the hepatopancreas. During hypo-osmotic stress, amino acid oxidation does not seem to be one of the pathways implicated in the decrease of the amino acid pools in hepatopancreas and jaw muscle. In contrast, during hyperosmotic stress the reduction in ¹⁴C-alanine oxidation appears to be one of the mechanisms involved in the increase of the amino acid pool in the hepatopancreas.     

Regulation of Cl− Secretion by Extracellular ATP in Cultured Mouse Endometrial Epithelium

The present study explored regulation of electrogenic ion transport across cultured mouse endometrial epithelium by extracellular ATP using the short-circuit current (I _SC ) and the patch-clamp techniques. The cultured endometrial monolayers responded to apical application of ATP with an increase in I _SC in a concentration-dependent manner (EC₅₀ at 3 μm). Replacement of Cl⁻ in the bathing solution or treatment of the cells with Cl⁻ channel blockers, DIDS and DPC, markedly reduced the I _SC , indicating that a substantial portion of the ATP-activated I _SC was Cl⁻-dependent. Amiloride at a concentration (10 μm) known to block Na⁺ channels was found to have no effect on the ATP-activated I _SC excluding the involvement of Na⁺ absorption. Adenosine was found to have little effect on the I _SC excluding the involvement of P₁ receptors. The effect of UTP, a potent P_2U receptor agonist on the I _SC was similar to that of ATP while potent P_2X agonist, α-β-Methylene adenosine 5′-triphosphate (α-β-M-ATP) and P_2Y agonist, 2-methylthio-adenosine triphosphate (2-M-ATP), were found to be ineffective. The effect of ATP on I _SC was mimicked by the Ca²⁺ ionophore, ionomycin, indicating a role of intracellular Ca²⁺ in mediating the ATP response. Confocal microscopic study also demonstrated a rise in intracellular Ca²⁺ upon stimulation by extracellular ATP. In voltage-clamped endometrial epithelial cells, ATP elicited a whole-cell Cl⁻ current which exhibited outward rectification and delayed activation and inactivation at depolarizing and hyperpolarizing voltages, respectively. The results of the present study demonstrate the presence of a regulatory mechanism involving extracellular ATP and P_2U purinoceptors for endometrial Cl⁻ secretion.     

Temperature and starvation affect the hemolymph acid-base balance of the xeric yellow scorpion,Leiurus quinquestriatus

Summary Water conservation is crucial for terrestrial animals such as scorpions which generally live in xeric habitats. One route of water loss is evaporation from their book lungs. In order to save water, scorpions may have a high resistance to gas exchange with the environment. If this is so then the partial pressure of CO₂ in their hemolymph, PCO₂, must be high. Does this affect their acid-base balance? Hemolymph PCO₂ and pH in normally-fed or starved desert-dwelling yellow scorpions Leiurus quinquestriatus were studied in vivo as functions of temperature. An ambient temperature increase (lasting at least 3 days) resulted in a rise of PCO₂ and a fall of pH, with thermal coefficients of 1.6 Torr · °C^-1 and-0.016 pH unit · °C^-1, respectively. The thermal coefficients for cell-free hemolymph studied in vitro were the same. At 28 °C, 3–6 weeks of starvation led to a 4.8 Torr increase in PCO₂ and a 0.056 unit decrease in pH. The in vivo PCO₂ values are among the highest, and pH values are the lowest of the terrestrial arthropods studied so far, e.g., at 28 °C they are 29 Torr and 7.15 pH respectively. It is argued that this particular acid-base balance with a marked hypercapnia is typical of a successful xeric air-breathing animal.Abbreviations ABB acid-base balance - PCO₂ partial pressure of CO₂ - PO₂ partial pressure of O₂     

Active transport of CO2 by three species of marine microalgae

The occurrence of an active CO₂ transport system and of carbonic anhydrase (CA) has been investigated by mass spectrometry in the marine, unicellular rhodophyte Porphyridium cruentum (S.F. Gray) Naegeli and two marine chlorophytes Nannochloris atomus Butcher and Nannochloris maculata Butcher. Illumination of darkened cells incubated with 100 μM H¹³CO₃^? caused a rapid initial drop, followed by a slower decline in the extracellular CO₂ concentration. Addition of bovine CA to the medium raised the CO₂ concentration by restoring the HCO₃^?–CO₂ equilibrium, indicating that cells were taking up CO₂ and were maintaining the CO₂ concentration in the medium below its equilibrium value during photosynthesis. Darkening the cell suspensions caused a rapid increase in the extracellular CO₂ concentration in all three species, indicating that the cells had accumulated an internal pool of unfixed inorganic carbon. CA activity was detected by monitoring the rate of exchange of ¹⁸O from ¹³C¹⁸O₂ into water. Exchange of ¹⁸O was rapid in darkened cell suspensions, but was not inhibited by 500 μM acetazolamide, a membrane‐impermeable inhibitor of CA, indicating that external CA activity was not present in any of these species. In all three species, the rate of exchange was completely inhibited by 500 μM ethoxyzolamide, a membrane‐permeable CA‐inhibitor, showing that an intracellular CA was present. These results demonstrate that the three species are capable of CO₂ uptake by active transport for use as a carbon source for photosynthesis.     

β2 adrenoceptor signaling regulates ion transport in 16HBE14o- human airway epithelial cells

We investigated the regulation of Cl⁻ secretion by adrenoceptors in polarized 16HBE14o- human bronchial epithelial cells. Treatment with the nonselective β adrenoceptor agonist isoprenaline stimulated an increase in short-circuit current (I_SC), which was inhibited by the β adrenoceptor blocker propranolol. Treatment with procaterol, an agonist specific for the β₂ adrenoceptor subtype, stimulated a similar increase in I_SC, which was inhibited by the β₂ adrenoceptor antagonist ICI 118551. Inhibitors of cystic fibrosis transmembrane conductance regulator (CFTR) and calcium-activated Cl⁻ channel (CaCC), but not K⁺ channel blockers, were able to inhibit the increase in I_SC. “Trimultaneous” recording of I_SC and intracellular cyclic adenosine monophosphate (cAMP) and Ca²⁺ levels in 16HBE14o- epithelia confirmed that the I_SC induced by isoprenaline or procaterol involved both cAMP and Ca²⁺ signaling. Our results demonstrate that β₂ adrenoceptors regulate Cl⁻ secretion in the human airway epithelium by activating apical CFTRs and CaCCs via cAMP-dependent and intracellular Ca²⁺-dependent mechanisms, respectively.     

Physiological and Biochemical Characteristics of Sugar Beet Plants Grown at an Increased Carbon Dioxide Concentration and at Various Nitrate Doses

Three-week-old sugar beet (Beta vulgaris L.) seedlings were grown for an additional four weeks under controlled conditions: in river sand watered with a modified Knop mixture containing one half-fold (0.5N), standard (1N), and or threefold (3N) nitrate amount, at the irradiance of 90 W/m² PAR, and at the carbon dioxide concentrations of 0.035% (1C treatment) or 0.07% (2C treatment). The increase in the carbon dioxide concentration and in the nitrogen dose resulted in an increase in the leaf area and the leaf and root dry weight per plant. With the increase in the nitrogen dose, morphological indices characterizing leaf growth increased more noticeably in 1C plants than in 2C plants. And vice versa, the effects of increased CO₂ concentration were reduced with the increase in the nitrogen dose. Roots responded to the changes in the CO₂ and nitrate concentrations otherwise than leaves. At a standard nitrate dose (1N), the contents of proteins and nonstructural carbohydrates (sucrose and starch) in leaves depended little on the CO₂ concentration. At a double CO₂ concentration, the content of chlorophyll somewhat decreased, and the net photosynthesis rate (P _n) calculated per leaf area unit increased. An increase in the nitrogen dose did not affect the leaf carbohydrate content of the 1C and 2C plants except the leaves of the 2C-3N plants, where the carbohydrate content decreased. In 1C and 2C plants, an increase in the nitrogen dose caused an increase in the protein and chlorophyll content. Specific P _n values somewhat decreased in 1C-0.5N plants and had hardly any dependence on the nitrate dose in the 2C plants. The carbohydrate content in roots did not depend on the CO₂ concentration, and the content was the highest at 0.5N. Characteristic nitrogen dose-independent acclimation of photosynthesis to an increased carbon dioxide concentration, which was postulated previously [1], was not observed in our experiments with sugar beet grown at doubled carbon dioxide concentration.     

Sustained effects of atmospheric [CO2] and nitrogen availability on forest soil CO2 efflux

Soil CO₂ efflux (F_soil) is the largest source of carbon from forests and reflects primary productivity as well as how carbon is allocated within forest ecosystems. Through early stages of stand development, both elevated [CO₂] and availability of soil nitrogen (N; sum of mineralization, deposition, and fixation) have been shown to increase gross primary productivity, but the long‐term effects of these factors on F_soil are less clear. Expanding on previous studies at the Duke Free‐Air CO₂ Enrichment (FACE) site, we quantified the effects of elevated [CO₂] and N fertilization on F_soil using daily measurements from automated chambers over 10 years. Consistent with previous results, compared to ambient unfertilized plots, annual F_soil increased under elevated [CO₂] (ca. 17%) and decreased with N (ca. 21%). N fertilization under elevated [CO₂] reduced F_soil to values similar to untreated plots. Over the study period, base respiration rates increased with leaf productivity, but declined after productivity saturated. Despite treatment‐induced differences in aboveground biomass, soil temperature and water content were similar among treatments. Interannually, low soil water content decreased annual F_soil from potential values – estimated based on temperature alone assuming nonlimiting soil water content – by ca. 0.7% per 1.0% reduction in relative extractable water. This effect was only slightly ameliorated by elevated [CO₂]. Variability in soil N availability among plots accounted for the spatial variability in F_soil, showing a decrease of ca. 114 g C m^?2 yr^?1 per 1 g m^?2 increase in soil N availability, with consistently higher F_soil in elevated [CO₂] plots ca. 127 g C per 100 ppm [CO₂] over the +200 ppm enrichment. Altogether, reflecting increased belowground carbon partitioning in response to greater plant nutritional needs, the effects of elevated [CO₂] and N fertilization on F_soil in this stand are sustained beyond the early stages of stand development and through stabilization of annual foliage production.