Ethacrynic acid and furosemide alter Cl,K, and Na distribution between blood,choroid plexus,CSF, and brain

Can loop diuretics like ethacrynic acid and furosemide, when administered intravenously, significantly alter ion transport and fluid dynamics in CNS? To shed light on this unresolved issue, we tested the ability of these agents to effect redistribution of Na, K and Cl in adult rat brain. Cl penetration into various CNS regions was assessed as the volume of distribution, i.e., uptake, of³⁶Cl from blood. Ethacrynic acid and furosemide (50 mg/kg IV) reduced by 20–30% the rate of permeation of³⁶Cl across the blood-CSF barrier, and they elevated [K] and [Cl] in choroid plexus (CP) by 15–25%. The loop diuretic-induced buildup of K and Cl in CP (lateral and 4th ventricle) was likely a reflection of decreased movement of these ions across the apical membrane into CSF.³⁶Cl activity in parietal cortex and pons-medulla decreased in treatment with furosemide and ethacrynic acid, due to slowing of Cl transport across blood-brain and/or blood-CSF barriers. Our inhibitory findings in intact rats are consistent with those from previous in vitro experiments demonstrating diminution by loop diuretics of Na, K and Cl transport across isolated CP membranes.     

Uptake of 36Cl and 22Na by the Choroid Plexus-Cerebrospinal Fluid System: Evidence for Active Chloride Transport by the Choroidal Epithelium

Abstract: Cl and Na transport by the lateral ventricle (LVCP) and fourth ventricle (4VCP) choroid plexuses were examined by kinetic analysis of ³⁶Cl and ²²Na uptake into the choroid plexus-CSF system of the adult rat. Both radioisotopes required more than 5 h to reach steady-state distribution in the in vivo choroid plexuses and CSF after intraperitoneal injection. Whereas the LVCP and 4VCP ³⁶Cl steady-state spaces were comparable (55–56%), the 4VCP ²²Na space (39%) tended to be greater than the LVCP ²²Na space (36%). No evidence for inexchangeable Cl or Na was found for the choroid plexuses; the radioisotopic and chemical spaces were not significantly different. Choroid plexus ³⁶Cl and ²²Na uptake curves were resolved into two components, a fast component ( t _1/2 0.02–0.05 h) and a slow component ( t _1/2 0.85–1.93 h). By analysis of the distribution of [³H]inulin, [³H]mannitol, and ⁵¹Cr-tagged erythrocytes within the choroid plexuses, the fast component of ³⁶Cl and ²²Na uptake was found to represent extracellular and erythrocyte contributions to the tissue radioactivity, whereas the slow component represented isotope movement into the epithelial cell compartment. The calculated cell [Cl] of LVCP and 4VCP, 67 mmol/kg cell water, was 3.9 times greater than that predicted by the membrane potential for passive distribution. It is postulated that Cl is actively transported into the choroid epithelial cell across the basolateral membrane; the energy source for active Cl transport may be the Na electrochemical potential gradient (˜90 mV), which is twice that of the Cl electrochemical potential gradient (˜45 mV).     

Blood–Cerebrospinal Fluid and Blood-Brain Barriers Imaged by 18F-Labeled Metabolites of 18F-Setoperone Studied in Humans Using Positron Emission Tomography

18F-Setoperone, a sensitive radioligand for brain serotonin 5-HT2 receptor positron emission tomography studies, is metabolized into 18F-labeled metabolites, which participate in blood 18F radioactivity. Its main metabolite, identified as reduced 18F-setoperone, was synthesized and studied in humans to determine if 18F-labeled metabolites of 18F-setoperone (a) enter into the brain, (b) bind to the 5-HT2 receptor, and (c) explain the increase of 18F radioactivity in the free fraction in blood measured following 18F-setoperone injection. After reduced 18F-setoperone injection, the brain-to-blood 18F radioactivity concentration ratio (a) was low, at the beginning, indicating that this metabolite did not cross the blood-brain barrier; (b) was increased thereafter, with a higher radioactivity level in the choroid plexus than in brain tissue, suggesting a blood-CSF barrier crossing due to radioligand hydrophilicity; and (c) showed similar kinetics for cerebellum and frontal cortex, indicating that radioactive metabolites of 18F-setoperone did not bind to the 5-HT2 receptor. Because hydrophilic 18F-labeled metabolites of 18F-setoperone increased 18F radioactivity in the free fraction in blood, we quantified the relation between 18F-setoperone metabolism and free fraction kinetics in blood. A significant negative correlation was found between metabolism and free fraction rate constants in blood, showing it was possible to predict the 18F-setoperone metabolism rate using free fraction kinetics in blood. This will allow us to avoid the use of radio-TLC, a reference method that is difficult to use when multiple samples must be analyzed. A hydrophilic positron-emitter radioligand could also be used to study the blood-CSF barrier.     

Diurnal variation of Δ13CO2, ΔC18O16O and evaporative site enrichment of δH218O in Piper aduncum under field conditions in Trinidad

Concurrent measurements of gas exchange, instantaneous isotope discrimination (Δ) against ¹³CO₂ and C¹⁸O¹⁶O, and extent of ¹⁸O enrichment in H₂O at the evaporative sites, were followed in a tropical forest pioneer, Piper aduncum, on two different days in Trinidad during February 1995. Δ¹³CO₂ differed from that predicted from measurements of internal:external CO₂ concentration (C_i/C_a) and showed a wide range of values which decreased throughout the course of the day. Derivation of C_c (the CO₂ concentration at the carboxylation site) was not possible using carbon isotope discrimination under field conditions in situ and was derived assuming a constant value of internal transfer conductance (g_w). Under low rates of assimilation the derived C_c/C_a, like C_i/C_a, remained relatively stable over the course of both days and ΔC¹⁸O¹⁶O followed evaporative demand. Lower values of ΔC¹⁸O¹⁶O on day 2 occurred in response to the indirect effect of increased leaf-to-air vapour pressure deficits (VPD) and reduced stomatal conductance. For the first time, direct determination of the δH₂¹⁸O of transpired water vapour (δ_t) allowed derivation of evaporative site enrichment without the prerequisite of isotopic steady state (ISS) defined in the Craig and Gordon model. Generally, δ_t was less enriched than the source water (δ_s) in the morning and more enriched in the afternoon, which would be predicted from an increase and decrease in ambient VPD, respectively. On both days, leaves of P. aduncum approached ISS (indicated where δ_t≈δ_s) between 1300 and 1500 h. Evaporative site enrichment was maintained into the late afternoon, despite a decrease in ambient VPD. The data presented provide a greater insight into the natural variation in isotopic discrimination under field conditions, which may help to refine models of terrestrial biome discrimination.     

Passage of a δ-Opioid Receptor Selective Enkephalin, [d-Penicillamine2,5]Enkephalin, Across the Blood-Brain and the Blood-Cerebrospinal Fluid Barriers

Abstract: [ d -Penicillamine^2,5]enkephalin (DPDPE) is an enzymatically stable, δ-opioid receptor-selective peptide, which produces analgesia when given intracerebroventricularly. However, because only modest analgesic effects were seen after subcutaneous administration of DPDPE, it has been inferred that it does not cross the blood-brain barrier well. In this present study, a vascular brain perfusion technique in anesthetized rats was used to measure directly whether [³H]DPDPE could cross the blood-brain and/or the blood-CSF barriers. The results indicated that the brain uptake of [³H]DPDPE was significantly greater than that of [¹⁴C]sucrose, a vascular marker ( p < 0.01), and than that of [³H]DPDPE into the CSF ( p < 0.01). Furthermore, HPLC analysis confirmed the integrity of the ³H to DPDPE and demonstrated that intact [³H]DPDPE entered the brain. Although 1 m M leucine-enkephalin failed to inhibit uptake of [³H]DPDPE, unlabeled DPDPE (100 µ M ) caused a significant inhibition of the brain uptake ( p < 0.01) but not the CSF uptake of [³H]DPDPE. These data provide evidence that intact [³H]DPDPE enters the CNS of anesthetized rats by saturable and nonsaturable mechanisms. In addition, the saturable mechanism is likely to be found at the blood-brain barrier, with the blood-CSF barrier playing only a minor role in the brain uptake of this peptide.     

Correlating δ13C and δ18O in cellulose of trees

We measured the carbon and oxygen isotopic composition of stem cellulose of Pinus sylvestris, Picea abies, Fagus sylvatica and Fraxinus excelsior. Several sites along a transect of a small valley in Switzerland were selected which differ in soil moisture conditions. At every site, six trees per species were sampled, and a sample representing a mean value for the period from 1940 to 1990 was analysed. For all species, the mean site δ¹³C and δ¹⁸O of stem cellulose are related to the soil moisture availability, whereby higher isotope ratios are found at drier sites. This result is consistent with isotope fractionation models when assuming enhanced stomatal resistance (thus higher δ¹³C of incorporated carbon) and increased oxygen isotope enrichment in the leaf water (thus higher δ¹⁸O) at the dry sites. δ¹⁸ O-δ¹³C plots reveal a linear relationship between the carbon and oxygen isotopes in cellulose. To interpret this relationship we developed an equation which combines the above-mentioned fractionation models. An important new parameter is the degree to which the leaf water enrichment is reflected in the stem cellulose. In the combined model the slope of the δ¹⁸O-δ¹³C plot is related to the sensitivity of the p_i/p_a of a plant to changing relative humidity.     

Soil 13C–15N dynamics in an N2-fixing clover system under long-term exposure to elevated atmospheric CO2

Reduced soil N availability under elevated CO₂ may limit the plant's capacity to increase photosynthesis and thus the potential for increased soil C input. Plant productivity and soil C input should be less constrained by available soil N in an N₂‐fixing system. We studied the effects of Trifolium repens (an N₂‐fixing legume) and Lolium perenne on soil N and C sequestration in response to 9 years of elevated CO₂ under FACE conditions. ¹⁵N‐labeled fertilizer was applied at a rate of 140 and 560 kg N ha^?1 yr^?1 and the CO₂ concentration was increased to 60 Pa pCO₂ using ¹³C‐depleted CO₂. The total soil C content was unaffected by elevated CO₂, species and rate of ¹⁵N fertilization. However, under elevated CO₂, the total amount of newly sequestered soil C was significantly higher under T. repens than under L. perenne. The fraction of fertilizer‐N (f_N) of the total soil N pool was significantly lower under T. repens than under L. perenne. The rate of N fertilization, but not elevated CO₂, had a significant effect on f_N values of the total soil N pool. The fractions of newly sequestered C (f_C) differed strongly among intra‐aggregate soil organic matter fractions, but were unaffected by plant species and the rate of N fertilization. Under elevated CO₂, the ratio of fertilizer‐N per unit of new C decreased under T. repens compared with L. perenne. The L. perenne system sequestered more ¹⁵N fertilizer than T. repens: 179 vs. 101 kg N ha^?1 for the low rate of N fertilization and 393 vs. 319 kg N ha^?1 for the high N‐fertilization rate. As the loss of fertilizer‐¹⁵N contributed to the ¹⁵N‐isotope dilution under T. repens, the input of fixed N into the soil could not be estimated. Although N₂ fixation was an important source of N in the T. repens system, there was no significant increase in total soil C compared with a non‐N₂‐fixing L. perenne system. This suggests that N₂ fixation and the availability of N are not the main factors controlling soil C sequestration in a T. repens system.     

THE UPTAKE OF 36Cl INTO ASTROCYTES IN TISSUE CULTURE BY A POTASSIUM-DEPENDENT, SATURABLE PROCESS

Abstract— Previous suggestions that the K⁺-dependent uptake of chloride by mammalian cerebral cortex is localized to cortical astrocytes has been investigated in two lines of neural cells in tissue culture. Hamster astrocytes and mouse neuroblastoma cells were each cultured on small glass cover slips in Eagle's (astrocytes) or Dulbecco's (neuroblastoma) supplemented media. At 48 h, cultures were incubated at 37°C for 1–60 min in the presence of ³⁶Cl plus [³H]inulin (as a measure of extracellular space). In media containing approximately 140 mm chloride, the rate of uptake of ³⁶Cl by the astrocytes was a function of the concentration of K⁺ (range 5–54 mm ) in the medium, and the uptake was characterized by saturation kinetics and an apparent K_m of 38·5 mm . The uptake was temperature and apparently energy dependent, significantly inhibited by 5 or 10 mm Br^-, I^-, SCN^- or ClO₄^-, and competitively inhibited by 10 mm acetazolamide (K_i= 27·1 mm ). None of these characteristics were observed with neuroblastoma cultures studied under similar conditions. In chloride-free media, the cellular K/Na ratio of the astrocytes shifted from the usual value of 4·55 to a value of 0·29, the culture medium became more alkaline than normal, and the cells spontaneously sloughed from the cover slips but remained normally viable. Our observations are the first to provide direct support for previous suggestions that there is a mediated, K⁺-dependent coupled cation, chloride and fluid uptake by mammalian cerebrocortical glia and that this uptake is an enzymatically catalysed process. The observations have been discussed in terms of a presumed central role for astrocytes in modulating the external ionic milieu of the neurons they surround and in terms of implications for epilepsy, stroke and cortical edema.     

Permeability of the blood cerebrospinal fluid barrier during acute immune complex disease

Changes in the permeability of the blood-brain barrier (BBB) and blood-cerebrospinal fluid (blood-CSF) barrier in rabbits were assessed by using a sensitive double isotope technique at different times after the induction of acute immune complex disease (AICD). Induction of AICD was done with a single large dose of bovine serum albumin, whereas controls received only saline. Animals were sacrificed 6, 9, 12, 15, and 18 days after induction. Extravasation of protein was measured by injecting rabbits i.v. with 131I-rabbit serum albumin (RSA) 24 hr before sacrifice. In order to correct for intravascular blood volume, 125I-RSA was injected 5 min before sacrifice. Extravascular blood equivalents (EVBE), a measure of barrier permeability, were elevated in the CSF of rabbits sacrificed on days 12 and 15. None of the brain regions from any of the animal groups showed any changes or significant differences from controls in EVBE values on these days. These results indicate that there was an increase in the permeability of the blood-CSF barrier to radiolabeled albumin but not in the BBB proper during the time that CSF IgG levels were elevated in AICD. The potential significance of these findings for the mechanisms mediating central nervous system involvement in systemic lupus erythematosus is discussed.     

Control of sodium permeability of the outer barrier in toad skin

Summary The²⁴Na efflux (J _eff ^Na ) (i.e., the rate of appearance of²⁴Na in the outer compartment) in the isolated short-circuited toad skin bathed by NaCl-Ringer's solution on both sides is composed of para- and transcellular components of almost equal magnitudes. This relies on the assumption that amiloride acts on the transcellular component only and could block it completely.Ouabain induces a large transient increase of the transcellular component. This increase, which starts within a few minutes after the addition of ouabain, is due to electrical depolarization of the outer barrier, rather than a consequence of blocking Na recirculation across the inner barrier. The subsequent decline ofJ _eff ^Na , which takes place after the ouabain-inducedJ _eff ^Na peak, is due to a progressive block of outer barrier Na channels with time, which can eventually be complete, depending on the duration of action of ouabain. As the external Na concentration was always kept high and constant in these experiments, the results indicate that a rise in cell Na concentration, and not in the outer bathing solution, is the signal that triggers the reduction of outer barrier Na permeability (P ₀ ^Na ).Ouabain has no effect uponJ _eff ^Na with Na-free solution bathing the outer and NaCl-Ringer's solution the inner skin surface, showing the importance of Na penetration across the outer barrier, and not across the inner barrier due to its low Na permeability, in the process of closing the Na channels of this structure.Step changes from Na 115mm to Na-free external solution, or vice-versa, may affect both the outer barrier electrical potential difference (PD₀) and cell Na concentration (Na) _c . Therefore, the behavior ofJ _eff ^Na depends on which variable (if PD₀ or (Na) _c regulated outer barrier Na permeability) is most affected by step changes in outer bathing solution Na concentration.Amiloride in the control condition blocks the transcellular component ofJ _eff ^Na . However, in the condition of approximate short-circuiting of the outer barrier and high cellular Na concentration induced by long term effects of ouabain, when the Na channels of the outer barrier are already blocked by elevated cell Na concentration, amiloride may induce the opposite effect, increasing Na permeability of the outer barrier.With outer barrier Na channels completely blocked by high cell Na concentration, PCMB in the outer bathing medium induces a large increase ofJ _eff ^Na , rendering these channels again amiloride sensitive.The results are consistent with the notion that Na efflux from cell compartment to the outer bathing solution goes through the amiloride-sensitive Na channels of the apical border of the superficial cell layer of toad skin, with an apparent Na permeability modulated by cell ionic environment, most probably the cell Na concentration.The ensemble of the present results are consistent with Na permeability regulation taking place at the outer barrier level. However, this precise location could only be made unambiguously by measurements across the individual outer cell membranes.     

The utilization of pre-anthesis reserves in grain filling of wheat. Assessment by steady-state 13CO2/12CO2 labelling

δ, C isotope composition relative to Pee Dee Belemnite
WSC, water-soluble carbohydrates
N, nitrogen
C, carbon
cv, cultivar
ME, efficiency of mobilized pre-anthesis C utilization in grain filling (g C g^–1C)

Significant mobilization of protein and carbohydrates in vegetative plant parts of wheat regularly occurs during grain filling. While this suggests a contribution of reserves to grain filling, the actual efficiency of mobilized assimilate conversion into grain mass (ME) is unknown. In the present study the contribution of pre-anthesis C (C fixed prior to anthesis) to grain filling in main stem ears of two spring wheat (Triticum aestivum L.) cultivars was determined by ¹³C/¹²C steady-state labelling. Mobilization of pre-anthesis C in vegetative plant parts between anthesis and maturity, and the contributions of water-soluble carbohydrates (WSC) and protein to pre-anthesis C mobilization were also assessed. Experiments were performed with two levels of N fertilizer supply in each of 2 years. Pre-anthesis reserves contributed 11–29% to the total mass of C in grains at maturity. Pre-anthesis C accumulation in grains was dependent on both the mass of pre-anthesis C mobilized in above-ground vegetative plant parts (r² = 0·87) and ME (defined as g pre-anthesis C deposited in grains per g pre-anthesis C mobilized in above-ground vegetative plant parts; r² = 0·40). ME varied between 0·48 and 0·75. The effects of years, N fertilizer treatments and cultivars on ME were all related to differences in the fractional contribution of WSC to pre-anthesis C mobilization. Multiple regression analysis indicated that C from mobilized pre-anthesis WSC may be used more efficiently in grain filling than C present in proteins at anthesis and mobilized during grain filling. Possible causes for variability of ME are discussed.     

Lobeline Displaces [3H]Dihydrotetrabenazine Binding and Releases [3H]Dopamine from Rat Striatal Synaptic Vesicles: Comparison with d-Amphetamine

Abstract: Lobeline, an alkaloid from Indian tobacco (Lobelia inflata), is classified as a nicotinic agonist and is currently used as a smoking cessation agent. However, our previous in vitro studies demonstrate that lobeline does not act as a nicotinic agonist but alters presynaptic dopamine (DA) storage by potently inhibiting DA uptake into synaptic vesicles. Recently, d-amphetamine has been reported to act at the level of the synaptic vesicle to alter presynaptic function. The present in vitro studies further elucidate the mechanism of lobeline's action and compare its effects with those of d-amphetamine. [³H]Dihydrotetrabenazine ([³H]DTBZ), used routinely to probe a high-affinity binding site on the vesicular monoamine transporter (VMAT2), bound to vesicle membranes from rat striatum with a K_D of 1.67 nM and B_max of 8.68 pmol/mg of protein. Lobeline inhibited [³H]DTBZ binding with an IC₅₀ of 0.90 µM, consistent with its previously reported IC₅₀ of 0.88 µM for inhibition of [³H]DA uptake into vesicles. These results suggest that lobeline specifically interacts with DTBZ sites on VMAT2 to inhibit DA uptake into synaptic vesicles. Interestingly, d-amphetamine inhibited [³H]DTBZ binding to vesicle membranes with an IC₅₀ of 39.4 µM, a concentration 20 times greater than reported for inhibition of VMAT2 function, suggesting that d-amphetamine interacts with a different site than lobeline on VMAT2 to inhibit monoamine uptake. Kinetic analysis of [³H]DA release from [³H]DA-preloaded synaptic vesicles in the absence of drug revealed a t_1/2 of 2.12 min. Lobeline and d-amphetamine evoked [³H]DA release with EC₅₀ values of 25.3 and 2.22 µM, respectively. At a concentration 10 times the EC₅₀, lobeline and d-amphetamine significantly decreased the t_1/2 of [³H]DA release to 1.58 and 1.48 min, respectively. Thus, in contrast to d-amphetamine, which is equipotent in inhibiting DA uptake and promoting release from the synaptic vesicles, lobeline more potently (28-fold) inhibits DA uptake (via an interaction with the DTBZ site on VMAT2) than it evokes DA release to redistribute presynaptic DA storage.     

Co-permeability of 3H-labelled water and 14C-labelled organic acids across isolated Prunus laurocerasus cuticles: effect of temperature on cuticular paths of diffusion

Co‐permeability of ³H‐labelled water and ¹⁴C‐labelled benzoic acid or 2,4‐dichlorophenoxyacetic acid across isolated cuticular membranes of Prunus laurocerasus L. was measured at temperatures ranging from 15 to 50 °C. The water and benzoic acid permeances were highly correlated over the whole temperature range investigated, whereas water and 2,4‐dichlorophenoxyacetic acid permeances were only correlated between 15 and 30 °C. The activation energies of cuticular permeability calculated from Arrhenius plots were 40 kJ mol^?1 for water and benzoic acid and 115 kJ mol^?1 for 2,4‐dichlorophenoxyacetic acid. The slopes of the Arrhenius plots of 2,4‐dichlorophenoxyacetic acid were linear between 15 and 50 °C, whereas pronounced phase transitions around 30 °C were observed for water and benzoic acid permeability. However, with isolated polymer matrix membranes, where cuticular waxes forming the transport‐limiting barrier of cuticles have been extracted, phase transitions were not observed for water and benzoic acid. It is concluded that temperatures above 30 °C caused structural changes in the transport‐limiting barrier of the cuticles leading to additional paths of diffusion for water and benzoic acid but not for 2,4‐dichlorophenoxyacetic acid.     

18O composition of CO2 and H2O ecosystem pools and fluxes in a tallgrass prairie: Simulations and comparisons to measurements

In this paper we describe measurements and modeling of ¹⁸O in CO₂ and H₂O pools and fluxes at a tallgrass prairie site in Oklahoma. We present measurements of the δ¹⁸O value of leaf water, depth‐resolved soil water, atmospheric water vapor, and Keeling plot δ¹⁸O intercepts for net soil‐surface CO₂ and ecosystem CO₂ and H₂O fluxes during three periods of the 2000 growing season. Daytime discrimination against C¹⁸OO, as calculated from measured above‐canopy CO₂ and δ¹⁸O gradients, is also presented. To interpret the isotope measurements, we applied an integrated land‐surface and isotope model (ISOLSM) that simulates ecosystem H₂¹⁸O and C¹⁸OO stocks and fluxes. ISOLSM accurately predicted the measured isotopic composition of ecosystem water pools and the δ¹⁸O value of net ecosystem CO₂ and H₂O fluxes. Simulations indicate that incomplete equilibration between CO₂ and H₂O within C₄ plant leaves can have a substantial impact on ecosystem discrimination. Diurnal variations in the δ¹⁸O value of above‐canopy vapor had a small impact on the predicted δ¹⁸O value of ecosystem water pools, although sustained differences had a large impact. Diurnal variations in the δ¹⁸O value of above‐canopy CO₂ substantially affected the predicted ecosystem discrimination. Leaves dominate the ecosystem ¹⁸O‐isoflux in CO₂ during the growing season, while the soil contribution is relatively small and less variable. However, interpreting daytime measurements of ecosystem C¹⁸OO fluxes requires accurate predictions of both soil and leaf ¹⁸O‐isofluxes.     

Intracellular Calcium Dynamics and Cellular Energetics in Ischemic NG108-15 Cells Studied by Concurrent 31P/19F and 23Na Double-Quantum Filtered NMR Spectroscopy

Abstract: The role of voltage-sensitive Ca²⁺ channels in mediating Ca²⁺ influx during ischemia was investigated in NG108-15 cells, a neuronal cell line that does not express glutamate-sensitive receptor-mediated Ca²⁺ channels. Concurrent ³¹P/¹⁹F and ²³Na double-quantum filtered (DQF) NMR spectra were used to monitor cellular energy status, intracellular [Ca²⁺] ([Ca²⁺]_i), and intracellular Na⁺ content in cells loaded with the calcium indicator 1,2-bis-(2-amino-5-fluorophenoxy)ethane-N,N,N′,N′-tetraacetic acid (5FBAPTA) during ischemia and reperfusion. Cells loaded with 5FBAPTA were indistinguishable from unloaded cells except for small immediate decreases in levels of phosphocreatine (PCr) and ATP. Ischemia induced a steady decrease in intracellular pH and PCr and ATP levels, and a steady increase in intracellular Na⁺ content; however, a substantial increase in [Ca²⁺]_i (about threefold) was seen only following marked impairment of cellular energy status, when PCr was undetectable and ATP content was reduced to 55% of control levels. A depolarization-induced increase in [Ca²⁺]_i could be completely blocked by 1 µM nifedipine, whereas up to 20 µM nifedipine had no effect on the increase in [Ca²⁺]_i seen during ischemia. These data demonstrate that voltage-gated Ca²⁺ channels do not mediate significant Ca²⁺ flux during ischemia in this cell line and suggest an important role for Ca²⁺_i stores, the Na⁺/Ca²⁺ antiporter, or other processes linked to cellular energy status in the increase in cytosolic Ca²⁺ level during ischemia.     

Nitrate and ammonium influxes in soybean (Glycine max) roots: direct comparison of 13N and 15N tracing

We compared influxes and internal transport in soybean plants (Glycine max cv. Kingsoy) of labelled N from external solutions where either ammonium or nitrate was labelled with the stable isotope¹⁵N and the radioactive isotope¹³N. The objective was to see whether mass spectrometric determinations of tissue ¹⁵N content were sufficiently sensitive to measure influxes accurately over short time periods. Our findings were as follows. (1) There was a close quantitative correspondence between estimates of N influx of individual plants using ¹⁵N or ¹³N measurements with either NO_3/^? or NH₄⁺ at 4 or 2 mol^?3, respectively in the external solution. (2) Transport to the shoot of N from NO₃ absorbed over a 5–15 min period could be monitored when the external NO₃^? concentration ranged from 0–05 to 4 mol m^?3. NH₄⁺ as the N source labelled shoot tissue more slowly, and estimates of the transport between root and shoot could be made only with ¹³N. (3) Influx of NO₃^? into root tissue could be measured by ¹⁵N enrichment after 5–10 min at concentrations approaching the probable K_M of the high-affinity transport system. (4) There was some indication of isotope discrimination, especially with respect to the movement of labelled N to the shoot, when NO₃^? is the N source. For many purposes, ¹⁵N tracing can be used satisfactorily to estimate influxes of both NO₃^? and NH₄⁺ in soybean roots. Use of the short-lived radio nuclide ¹³N remains the method of choice for more refined measurements of internal distribution and assimilation.     

Acquisition and within-plant allocation of 13C and 15N in CO2-enriched Quercus robur plants

We assessed the effects of doubling atmospheric CO₂ concentration, [CO₂], on C and N allocation within pedunculate oak plants (Quercus robur L.) grown in containers under optimal water supply. A short-term dual ¹³CO₂ and ¹⁵NO₃^? labelling experiment was carried out when the plants had formed their third growing flush. The 22-week exposure to 700 μl l^?1 [CO₂] stimulated plant growth and biomass accumulation (+53% as compared with the 350 μl l^?1 [CO₂] treatment) but decreased the root/shoot biomass ratio (-23%) and specific leaf area (-18%). Moreover, there was an increase in net CO₂ assimilation rate (+37% on a leaf dry weight basis; +71% on a leaf area basis), and a decrease in both above- and below-ground CO₂ respiration rates (-32 and -26%, respectively, on a dry mass basis) under elevated [CO₂]. ¹³C acquisition, expressed on a plant mass basis or on a plant leaf area basis, was also markedly stimulated under elevated [CO₂] both after the 12-h ¹³CO₂ pulse phase and after the 60-h chase phase. Plant N content was increased under elevated CO₂ (+36%), but not enough to compensate for the increase in plant C content (+53%). Thus, the plant C/N ratio was increased (+13%) and plant N concentration was decreased (-11%). There was no effect of elevated [CO₂] on fine root-specific ¹⁵N uptake (amount of recently assimilated ¹⁵N per unit fine root dry mass), suggesting that modifications of plant N pools were merely linked to root size and not to root function. N concentration was decreased in the leaves of the first and second growing flushes and in the coarse roots, whereas it was unaffected by [CO₂] in the stem and in the actively growing organs (fine roots and leaves of the third growth flush). Furthermore, leaf N content per unit area was unaffected by [CO₂]. These results are consistent with the short-term optimization of N distribution within the plants with respect to growth and photosynthesis. Such an optimization might be achieved at the expense of the N pools in storage compartments (coarse roots, leaves of the first and second growth flushes). After the 60-h ¹³C chase phase, leaves of the first and second growth flushes were almost completely depleted in recent ¹³C under ambient [CO₂], whereas these leaves retained important amounts of recently assimilated ¹³C (carbohydrate reserves?) under elevated [CO₂].     

Natural abundance of 13C and 15N in C3 and C4 vegetation of southern Africa: patterns and implications

The mean annual rainfall in southern Africa is found to explain over half of the observed variance in the stable nitrogen (N) isotopic signatures of C₃ vegetation in southern Africa (r²=0.54, P<0.01). The inverse relationship between the stable N isotopic signatures of foliar samples from C₃ vegetation and long‐term southern African rainfall is found on a scale larger than previously observed. A modest relationship is found between stable carbon (C) isotopic signatures of C₃ vegetation and rainfall across the region (r²=0.20, P<0.01). No such relationship is found between stable C and N isotopic signatures of C₄ vegetation and rainfall. The explanation of the relationship between ¹⁵N in C₃ vegetation and the mean annual rainfall presented here is that nutrient availability varies inversely with water availability. This suggests that water‐limited systems in southern Africa are more open in terms of nutrient cycling and therefore the resulting natural abundance of foliar ¹⁵N in these systems is enriched. The use of this relationship may be of value to those researchers modeling both the dynamics of vegetation and biogeochemistry across this region. The use of the isotopic enrichment in C₃ vegetation as a function of rainfall may provide an insight into nutrient cycling across the semi‐arid and arid regions of southern Africa. This finding has implications for the study of global change, especially as it relates to vegetation responses to changing regional rainfall regimes over time.     

On the Recovery of [3H]Noradrenaline from Different Metabolic Compartments of Rat Brain with Respect to the Role of Catechol-O-Methyltransferase

Abstract: Rats were treated with reserpine, desmethylimipramine, or carrier, either alone or in combination with tropolone. Either 10 min (t₁) or 1 h (t₂) after intraventricular injection of [³H]noradrenaline, they were decapitated. The total ³H activity and the recovery of [³H]noradrenaline were determined in tissue extracts from various brain regions. Maximum total ³H activity was measured at t₁ in all tropolone-treated rats; the mean sum of these results served as an estimate of the initial tissue concentration of [³H]noradrenaline. At t₁, 40–50% of the sum of [³H]noradrenaline and its metabolites was recovered unchanged in normal rats; reserpine and DMI reduced the recovery to 18–27%. In all groups, the decline of [³H]noradrenaline was retarded after t₁. Inhibition of catechol-O-methyltransferase by tropolone caused consistently elevated [³H]noradrenaline levels, but did not affect the metabolic rate after t₁ when compared with similarly pretreated, but tropolone-free rats. Thus, if catechol-O-methyltransferase was inhibited during the injection of [³H]noradrenaline, a higher percentage of the amine had been taken up into spaces with a slow noradrenaline turnover. The maximum increase was seen when the neuronal uptake, was inhibited by desmethylimipramine. This supported the hypothesis that an additional extraneuronal space exists, in addition to the known intraneuronal and extraneuronal compartments, which has a slow noradrenaline turnover. The tropolone effect on the noradrenaline recovery possibly shows that there might be a saturable “methylating system,” similar to that described for the periphery, in which catechol-O-methyltransferase is linked to the extraneuronal uptake₂. By affecting the access of noradrenaline to non-neuronal cells it might influence the rate of noradrenaline elimination from the intercellular space.     

δ13C of CO2 respired in the dark in relation to δ13C of leaf metabolites: comparison between Nicotiana sylvestris and Helianthus annuus under drought

The variations of δ¹³C in leaf metabolites (lipids, organic acids, starch and soluble sugars), leaf organic matter and CO₂ respired in the dark from leaves of Nicotiana sylvestris and Helianthus annuus were investigated during a progressive drought. Under well‐watered conditions, CO₂ respired in the dark was ¹³C‐enriched compared to sucrose by about 4‰ in N. sylvestris and by about 3‰ and 6‰ in two different sets of experiments in H. annuus plants. In a previous work on cotyledonary leaves of Phaseolus vulgaris, we observed a constant ¹³C‐enrichment by about 6‰ in respired CO₂ compared to sucrose, suggesting a constant fractionation during dark respiration, whatever the leaf age and relative water content. In contrast, the ¹³C‐enrichment in respired CO₂ increased in dehydrated N. sylvestris and decreased in dehydrated H. annuus in comparison with control plants. We conclude that (i) carbon isotope fractionation during dark respiration is a widespread phenomenon occurring in C₃ plants, but that (ii) this fractionation is not constant and varies among species and (iii) it also varies with environmental conditions (water deficit in the present work) but differently among species. We also conclude that (iv) a discrimination during dark respiration processes occurred, releasing CO₂ enriched in ¹³C compared to several major leaf reserves (carbohydrates, lipids and organic acids) and whole leaf organic matter.