Interstitial noradrenaline concentration of rat hearts as influenced by cellular catecholamine uptake mechanisms

Marked concentration differences of noradrenaline (NA) between the vascular and the interstitial compartment were detected by sampling interstitial transudate from isolated perfused rat hearts. The ratios of vascular/interstitial concentration amounted to 7.4 to 1.3 depending on the concentration of NA administered (3 × 10^–9 to 10^–6 M). These concentration differences were abolished by inhibitors of uptake₁ desipramine (DMI) I and uptake, (O-methyl-isoprenaline (OMI)). Neuronal uptake, was characterized by a K_m of 0.22 mol/l and a V_max of 370 pmol × min^–1 × gWWT^–1, extraneuronal uptake₂ by a K_UPTAKE of = 0.313 min^–4.The apparent permeability surface area (P×S)-product calculated from uptake rate and transcapillary concentration difference was significantly decreased by administrating 100 mol/l (NA) in presence of DMI. A presumed endothelial uptake mechanism contributing to catecholamine translocation was investigated in endothelial cells in culture. These cells showed a specific noradrenaline uptake with a K_m of 4.35 mol/l and a V_max of about 75 pmol × min^–1 x gWWT^–1. Any inhibiton by inhibitors of both of the two noradrenaline uptakes was lacking. The uptake rate of this mechanism is insufficient to contribute to the diffusive conductivity of the capillary wall (P × S-product). We conclude from our investigations on interstitial concentrations of catecholamines and transcapillary concentration differences, that the capillary wall, owing to its metabolic and diffusional characteristics, influences the exchange of catecholamines to a substantial and physiologically relevant extent.     

Omega-3 fatty acids and the peroxisome

The interactions between the omega-3 unsaturated fatty acids and peroxisomal function have been reviewed, in order to update and integrate knowledge in this area. Following a brief retrospective of the major clinical involvements of these fatty acids, the participation of the peroxisome in their metabolism has been appraised - the peroxisome being shown to exert a major influence on both the synthesis and degradation of the omega-3 fatty acids, with these effects flowing on to the widespread physiological implications of the derivative eicosanoids. Interactions between the omega-3 and omega-6 families of fatty acids have been discussed, as have the interdependent phenomena of peroxisome proliferation, membrane remodelling and cellular signalling. Amongst the signalling involvements covered were those of steroid hormone receptor superfamily, the phosphatidy1choline cycle, and the regulatory influences of oxygen free radicals. Comment has also been included on the separate biological roles of the individual omega-3 fatty acids, their influence on differential gene function, and on the molecular mechanisms of their pharmacological effects. It is concluded that the peroxisome is intimately involved in directing the metabolism and physiological influence of the omega-3 unsaturated fatty acids, and that this organelle merits much greater emphasis in future research aimed at unravelling the profound biological effects of these unique and multipotent compounds.     

Dietary folate affects the response of rats to nickel deprivation

Because vitamin B₁₂ and Ni are known to interact and because of the similar metabolic roles of vitamin B₁₂ and folate, an experiment was performed to determine the effect of dietary folate on Ni deprivation in rats. A 2×2 factorially arranged experiment used groups of nine weanling Sprague-Dawley rats. Dietary variables were Ni, as NiCl₂·6H₂O, 0 or 1 μg/g; and folic acid, 0 or 2 mg/kg. The basal diet, based on skim milk, contained less than 20 ng Ni/g. After 54 d, an interaction between dietary Ni and folate affected several variables including erythrocyte folate, plasma amino acids, and femur trace elements. For example, folate deprivation decreased erythrocyte folate; folate supplementation to the Ni-supplemented rats caused a larger increase in erythrocyte folate concentration than did folate supplementation to the Ni-deprived rats. Also, dietary Ni affected several plasma amino acids important in one-carbon metabolism (e.g., Ni deprivation increased the plasma concentrations of glycine and serine). This study shows that dietary Ni, folate, and their interaction can affect variables associated with one-carbon metabolism. This study does not show a specific site of action of Ni but it indicates that Ni may be important in processes related to the vitamin B₁₂-dependent pathway in methionine metabolism, possibly one-carbon metabolism. US Department of Agriculture, Agricultural Research Service, Northern Plans Area is an equal opportunity/affirmative action employer and all agency services are available without discrimination.     

Higher-plant chloroplast and cytosolic 3-phosphoglycerate kinases: a case of endosymbiotic gene replacement

Previous studies indicated that plant nuclear genes for chloroplast and cytosolic isoenzymes of 3-phosphoglycerate kinase (PGK) arose through recombination between a preexisting gene of the eukaryotic host nucleus for the cytosolic enzyme and an endosymbiont-derived gene for the chloroplast enzyme. We readdressed the evolution of eukaryotic pgk genes through isolation and characterisation of a pgk gene from the extreme halophilic, photosynthetic archaebacterium Haloarcula vallismortis and analysis of PGK sequences from the three urkingdoms. A very high calculated net negative charge of 63 for PGK from H. vallismortis was found which is suggested to result from selection for enzyme solubility in this extremely halophilic cytosol. We refute the recombination hypothesis proposed for the origin of plant PGK isoenzymes. The data indicate that the ancestral gene from which contemporary homologues for the Calvin cycle/glycolytic isoenzymes in higher plants derive was acquired by the nucleus from (endosymbiotic) eubacteria. Gene duplication subsequent to separation of Chlamydomonas and land plant lineages gave rise to the contemporary genes for chloroplast and cytosolic PGK isoenzymes in higher plants, and resulted in replacement of the preexisting gene for PGK of the eukaryotic cytosol. Evidence suggesting a eubacterial origin of plant genes for PGK via endosymbiotic gene replacement indicates that plant nuclear genomes are more highly chimaeric, i.e. contain more genes of eubacterial origin, than is generally assumed.Abbreviations PGK 3-phosphoglycerate kinase - FBA fructose-1,6-bisphosphate aldolase - GAPDH glyceraldehyde-3-phosphate dehydrogenase - TPI triosephosphate isomerase     

Changes in distribution of chloroplast adenylate kinase isoforms during floral induction

In the short day plant Chenopodium rubrum and the long day plant Nicotiana tabacum cv. Havana 425, adenylate kinase (EC 2.7.4.3) occurs as a family of isoforms, with at least two members localized in the chloroplast representing the main isoforms. In this work, isoforms were separated by anion exchange chromatography and relative isoform activities were compared between vegetative plants and plants induced to flowering. In both species examined, a light regime leading to floral induction resulted in a significant decrease in the activity of one chloroplast isoform. This decrease modified considerably the relative distribution of isoform activities, especially that between the two chloroplast activities.     

Short-term effects of urea amended with the urease inhibitor N-(n-butyl) thiophosphoric triamide on perennial ryegrass

The current study investigated the short-term physiological implications of plant nitrogen uptake of urea amended with the urease inhibitor N-(n-butyl) thiophosphoric triamide (nBTPT) under both greenhouse and field conditions. ¹⁵N labelled urea amended with 0.0, 0.01, 0.1 and 0.5% nBTPT (w/w) was surface applied at a rate equivalent to 100 kg N ha^–1 to perennial ryegrass in a greenhouse pot experiment. Root, shoot and soil fractions were destructively harvested 0.75, 1.75, 4, 7 and 10 days after fertilizer application. Urease activity was determined in each fraction together with ¹⁵N recovery and a range of chemical analyses. The effect of nBTPT amended urea on leaf tip scorch was evaluated together with the effect of the inhibitor applied on its own on plant urease activity.nBTPT-amended urea dramatically reduced shoot urease activity for the first few days after application compared to unamended urea. The higher the nBTPT concentration the longer the time required for shoot activity to return to that in the unamended treatment. At the highest inhibitor concentration of 0.5% shoot urease activity had returned to that of unamended urea by 10 days. Root urease activity was unaffected by nBTPT in the presence of urea but was affected by nBTPT in the absence of urea.Transient leaf tip scorch was observed approximately 7–15 days after nBTPT + urea application and was greatest with high concentrations of nBTPT and high urea-N application rates. New developing leaves showed no visual sign of tip necrosis.Urea hydrolysis of unamended urea was rapid with only 1.3% urea-N remaining in the soil after 1.75 days. N uptake and metabolism by ryegrass was rapid with ¹⁵N recovery from unamended urea, in the plant (shoot + root) being 33% after 1.75 days. Most of the ¹⁵N in the soil following the urea+0.5% nBTPT application was still as urea after 1.75 days, yet ¹⁵N plant recovery at this time was 25% (root+shoot). This together with other evidence, suggests that if urea hydrolysis in soil is delayed by nBTPT then urea can be taken up by ryegrass as the intact molecule, albeit at a significantly slower initial rate of uptake than NH₄ ⁺-N. Protein and water soluble carbohydrate content of the plant were not significantly affected by amending urea with nBTPT however, there was a significant effect on the composition of amino acids in the roots and shoots, suggesting a difference in metabolism.Although nBTPT-amended urea affected plant urease activity and caused some leaf-tip scorch the effects were transient and short-lived. The previously reported benefit of nBTPT in reducing NH₃ volatilization of urea would appear to far outweigh any of the observed short-term effects, as dry-matter production of ryegrass is increased.     

Hexose metabolism in discs excised from developing potato (Solanum tuberosum L.) tubers

Metabolism of radiolabelled hexoses by discs excised from developing potato (Solanum tuberosum L.) tubers was been investigated in the presence of acid invertase to prevent accumulation of labelled sucrose in the bathing medium (Viola, 1996, Planta 198: 179–185). When the discs were incubated with either [U-¹⁴C]glucose or [U-¹⁴C]fructose without unlabelled hexoses, the unidirectional rate of sucrose synthesis was insignificant compared with that of sucrose breakdown. The inclusion of unlabelled fructose in the medium induced a dramatic increase in the unidirectional rate of sucroses synthesis in the tuber discs. Indeed, the decline in the sucrose content observed when discs were incubated without exogenous sugars could be completely prevented by including 300 mM fructose in the bathing medium. On the other hand, the inclusion of unlabelled glucose in the medium did not significantly affect the relative incorporation of [U-¹⁴C]glucose to starch, sucrose or glycolytic products. Substantial differences in the intramolecular distribution of ¹³C enrichment in the hexosyl moieties of sucrose were observed when the discs were incubated with either [2-¹³C]fructose or [2-¹³C]glucose. The pattern of ¹³C enrichment distribution in sucrose suggested that incoming glucose was converted into sucrose via the sucrose-phosphate synthase pathway whilst fructose was incorporated directly into sucrose via sucrose synthase. Quantitative estimations of metabolic fluxes in vivo in the discs were also provided. The apparent maximal rate of glucose phosphorylation was close to the extractable maximum catalytic activity of glucokinase. On the other hand, the apparent maximal rate of fructose phosphorylation was much lower than the maximum catalytic activity of fructokinase, suggesting that the activity of the enzyme (unlike that of glucokinase) was regulated in vivo. Although in the discs incubated with or without fructose the rates of starch synthesis or glycolysis were similar, the relative partitioning of metabolic intermediates into sucrose was much higher in discs incubated with fructose (0.6% and 32.6%, respectively). It is hypothesised that the equilibrium of the reaction catalysed by sucrose synthase in vivo is affected in discs incubated with fructose as a result of the accumulation of the sugar in the tissue. This results in the onset of sucrose cycling. Incubation with glucose enhanced all metabolic fluxes. In particular, the net rate of starch synthesis increased from 2.0 mol · hexose · g FW^–1 · h^–1 in the absence of exogenous glucose to 3.7 mol · hexose · g FW^–1 · h^–1 in the presence of 300 mM glucose. These data are taken as an indication that the regulation of fructokinase in vivo may represent a limiting factor in the utilisation of sucrose for biosynthetic processes in developing potato tubers.Abbreviations ADPGlc adenosine 5-diphosphoglucose - Glc6P glucose-6-phosphate - hexose-P hexose phosphate - NMR nuclear magnetic resonance - UDPGlc uridine 5-diphosphoglucose Many thanks to L. Sommerville for skillfull assistance and to J. Crawford and J. Liu for useful discussions on flux analysis. The research was funded by the Scottish Office Agriculture and Fisheries Department.     

Generation of rhythmic and arrhythmic behaviour of Crassulacean acid metabolism in Kalanchoë daigremontiana under continuous light by varying the irradiance or temperature: Measurements in vivo and model simulations

Leaves of Kalanchoë daigremontiana Hamet et Perr. at a photon flux density (PFD) above 220 mol·m^–2s^–1 (400–700 nm) or at leaf temperatures above 27.0 °C showed a rapid loss of rhythmicity, and a more or less pronounced damping-out of the endogenous circadian rhythm of CO₂ exchange under continuous illumination. This rhythm was reinitiated after reduction of the PFD by 90–120 mol·m^–2·s^–1 or reduction of leaf temperature by 3.5–11.0 °C under otherwise unchanged external conditions. The reduction in the magnitude of the external control parameter of the Crassulacean acid metabolism (CAM) rhythm (i.e. PFD or leaf temperature) set the phase of the new rhythm. The maxima of CO₂ uptake occurred about 5, 28, 51, 75 h after the reduction. Simulations with a CAM model under comparable conditions showed a similar behaviour. The influence of temperature on the endogenous CAM rhythm observed in K. daigremontiana in vivo could be simulated by incorporating into the model temperature-dependent switch modes for passive efflux of malate from the vacuole to the cytoplasm. Thus, the model indicates that tonoplast function plays an important role in regulation of the endogenous CAM rhythm in K. daigremontiana.Abbreviations CAM Crassulacean acid metabolism - PAR photosynthetically active radiation - PFD photon flux density This work was supported by a grant to F.B. and U.L. from Teilprojekt B5 in the Sonderforschungsbereich 199 of the Deutsche Forschungsgemeinschaft (Bonn, Germany) and by a grant to T. E. E. G. from the Sudienstiftung des deutschen Volkes (Bonn, Germany). Erika Ball is thanked for processing of time-course data for the analysis of Fourier spectra.     

Anaerobic metabolism in the leech (Hirudo medicinalis L.): direct and indirect calorimetry during severe hypoxia

Anaerobic metabolism in the limnic annelid Hirudo medicinalis L. was investigated by direct and indirect calorimetry. During long-term severe hypoxia, the rate of heat dissipation was reduced up to 13% of the aerobic rate. At the same time, the rate of ATP turnover was reduced to about 30% of the aerobic rate, indicating that metabolic depression is an important mechanism to ensure survival of the leech during environmental anaerobiosis. Heat dissipation during hypoxia was monitored under two experimental conditions, favouring either concomitant hypocapnia (continuous N₂ bubbling) or hypercapnia (self-induced hypoxia). The reduction in heat dissipation during hypocapnic hypoxia was less pronounced than during hypercapnic hypoxia, indicating that the different experimental conditions may influence anaerobic metabolism and the extent of metabolic depression. Biochemical analysis of known anaerobic substrates and endproducts provided the basis for indirect calorimetry during self-induced hypoxia. From changes in metabolites, the expected heat dissipation was calculated for initial (0–8 h) and long-term severe hypoxia (8–72 h). During the initial period, the calculated heat dissipation fully accounted for direct calorimetric determination. During long-term hypoxia, only 71% of the measured heat production could be explained from biochemical analysis of metabolites. Therefore, an additional unknown endproduct cannot be excluded, especially when anaerobic ammonia production and analysis of the carbohydrate balance are considered.Abbreviations APW artificial pond water - HPLC high-performance liquid chromatography - fw fresh weight - HP heat production - HD heat dissipation - MR metabolic rate     

Sulfonates: novel electron acceptors in anaerobic respiration

The enrichment and isolation in pure culture of a bacterium, identified as a strain of Desulfovibrio, able to release and reduce the sulfur of isethionate (2-hydroxyethanesulfonate) and other sulfonates to support anaerobic respiratory growth, is described. The sulfonate moiety was the source of sulfur that served as the terminal electron acceptor, while the carbon skeleton of isethionate functioned as an accessory electron donor for the reduction of sulfite. Cysteate (alanine-3-sulfonate) and sulfoacetaldehyde (acetaldehyde-2-sulfonate) could also be used for anaerobic respiration, but many other sulfonates could not. A survey of known sulfate-reducing bacteria revealed that some, but not all, strains tested could utilize the sulfur of some sulfonates as terminal electron acceptor. Isethionate-grown cells of Desulfovibrio strain IC1 reduced sulfonate-sulfur in preference to that of sulfate; however, sulfate-grown cells reduced sulfate-sulfur in preference to that of sulfonate. Received: 2 May 1996 / Accepted: 8 June 1996