A daily study of the diatom spring bloom at Roscoff (France) in 1985. III. Free amino acids composition studied by HPLC analysis

The growth of a natural phytoplankton population was studiedduring a monospecific spring bloom of the diatom Rhizosoleniadelicatula at Roscoff (western English Channel). Direct examinationof the intracellular pool of 19 free amino acids (FAA) was usedas an index of the physiological status of the cells. TotalFAA in the particulate matter shows a general decrease duringthe bloom, and FAA cell content varies from 200 mM 1 cell volume^–1to 10 mM at the time of maximum biomass. FAA-N/particulate Nis <3% during the study, slightly decreasing at the timeof maximum biomass. Individual free amino acids appear reliablefor the development of the diatom biomass and good indicatorsof the growing population. At maximum biomass, major compoundsare glutamic acid (30.8 mol%), glutamine (11.7%), alanine (9.9%),isoleucine (6.6%) and lysine (6.4%). Serine, glycine, arginineand aspartic acid appear also as major components during otherperiods of the study. We suggest that the observed changes arean indication of the physiological state of the cells duringthe bloom. In particular glutamine (GLN), glutamic acid (GLU)and the GLN/GLU ratio allow the metabolic evolution of the naturalpopulation to be characterized. GLN is strictly linked to theliving biomass (Chla) while GLU is present at high percentagethroughout the study. The ecological significance of ß-alanine(BALA) is also revealed during this study, being strongly correlatedto degraded matter represented by phaeopigments. These resultsdemonstrate the validity of using individual free amino acidsas an aid in understanding the physiological status of algaein natural bloom conditions.     

Changes in intracellular and extracellular α-amino acids in Gloeothece during N2-fixation and following addition of ammonium

Changes in extracellular and intracellular free amino acids were followed during cyclic phases of N₂-fixation (acetylene reduction) by cultures of the axenic, non-heterocystous cyanobacterium Gloeothece incubated under alternating light and darkness or continuous illumination. Changes in intracellular amino acids were minor, with only arginine (increasing during N₂-fixation) and glutamate (decreasing during fixation) showing significant changes in cells incubated under 12 h light: 12 h dark. The intracellular concentration of glutamine in cultures was always very low and the value of the ratio glutamine: glutamate (GLN:GLU), used as an index of C–N status in eukaryote microbes, was consistently less than 0.05 suggesting that the cells were nitrogen-stressed. On addition of ammonium, there was a transient accumulation of intracellular glutamine, and the ratio GLN:GLU increased rapidly to a value greater than 0.5, typical of unstressed eukaryotes. In contrast to intracellular amino acids, there were significant changes in extracellular amino acids in cultures incubated under alternating light and darkness. Glycine, serine and alanine were released during the dark phase and were taken up again in the light, paralleling the diurnal pattern of nitrogenase activity (high in darkness). It is postulated that this release is usually retained in the mucilage surrounding the cells (but disturbed during even gentle filtration) and that this mucilage may constitute an extracellular vacuole.     

Glutamine's protection against cellular injury is dependent on heat shock factor-1

Glutamine (GLN) has been shown to protect cells, tissues, and whole organisms from stress and injury. Enhanced expression of heat shock protein (HSP) has been hypothesized to be responsible for this protection. To date, there are no clear mechanistic data confirming this relationship. This study tested the hypothesis that GLN-mediated activation of the HSP pathway via heat shock factor-1 (HSF-1) is responsible for cellular protection. Wild-type HSF-1 (HSF-1^+/+) and knockout (HSF-1^–/–) mouse fibroblasts were used in all experiments. Cells were treated with GLN concentrations ranging from 0 to 16 mM and exposed to heat stress injury in a concurrent treatment model. Cell viability was assayed with phenazine methosulfate plus tetrazolium salt, HSP-70, HSP-25, and nuclear HSF-1 expression via Western blot analysis, and HSF-1/heat shock element (HSE) binding via EMSA. GLN significantly attenuated heat-stress induced cell death in HSF-1^+/+ cells in a dose-dependent manner; however, the survival benefit of GLN was lost in HSF-1^–/– cells. GLN led to a dose-dependent increase in HSP-70 and HSP-25 expression after heat stress. No inducible HSP expression was observed in HSF-1^–/– cells. GLN increased unphosphorylated HSF-1 in the nucleus before heat stress. This was accompanied by a GLN-mediated increase in HSF-1/HSE binding and nuclear content of phosphorylated HSF-1 after heat stress. This is the first demonstration that GLN-mediated cellular protection after heat-stress injury is related to HSF-1 expression and cellular capacity to activate an HSP response. Furthermore, the mechanism of GLN-mediated protection against injury appears to involve an increase in nuclear HSF-1 content before stress and increased HSF-1 promoter binding and phosphorylation. knockout cells; amino acid; heat stress mechanism     

Effect of Si-status on diel variation of intracellular free amino acids in Thalassiosira weissflogii under low-light intensity

Twenty-one intracellular free amino acids were analysed during a 12-12 h light-dark cycle, on duplicate axenic cultures of Thalassiosira weissflogii (clone Actin, Provasoli-Guillard CCMP) under either Si-sufficient or Si-starved conditions. Total concentrations ranged between 40 and 165 fmol/cell. Total level as well as individual levels of amino acids decreased during the dark period, and GLN/GLU ratio was lower during the dark period. All these results were correlated with the light-dark carbon metabolism of the algae and related to the protein synthesis at night. The Si-starved cultures showed a lower total level of FAA compare to the Si-sufficient cultures, especially in the light period. Silica status of the cells affected more the metabolites of the dark respiration than the photorespiratory metabolites SER and GLY. Si deprivation induced higher range of ALA and VAL, and a decrease of the TCA metabolites GLU & ASP. Additionally, the relative percentage of ASP increased under Si starvation, at the expense of GLU, and this shift was emphasized in the dark period.     

Effects of 4-Aminopyridine on Extracellular Concentrations of Glutamate in Striatum of the Freely Moving Rat

4-aminopyridine (4-AP) is a voltage-sensitive K⁺-channel blocker extensively used in in vitro experiments as a depolarizing agent for the release of glutamate (GLU). This research investigated whether 4-AP could be used in in vivo experiments using microdyalisis. For that, the effects of 4-AP on the extracellular concentrations of glutamate (GLU), glutamine (GLN), taurine (TAU) and citrulline (CIT) in striatum of the freely moving rat were investigated. The effects of 4-AP were compared with those produced by perfusion with a high K⁺ (100 mM) medium. Intrastriatal perfusion with 4-AP (1, 5 and 10 mM) produced no effects on extracellular [GLU], [TAU] and [CIT], but decreased extracellular [GLN]. Perfusion with a high K⁺ (100 mM) medium increased extracellular [GLU] and [TAU], decreased extracellular [GLN], and had no effects on [CIT]. To test whether the lack of effects of 4-AP on extracellular [GLU] was due to GLU uptake mechanisms, 4-AP was perfused after a previous inhibition of GLU uptake with L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC). Under the effects of PDC (1 mM), 4-AP (1 mM) had no effects on extracellular [GLU], [TAU] and [CIT], but decreased extracellular [GLN]. These results show that 4-AP decreased extracellular [GLN] but failed to produce a significant release of GLU in striatum of the freely moving rat. Thus, 4-AP can not be used as a depolarizing agent for stimulating the release of GLU in in vivo studies using microdialysis.     

Intracellular Ca(2+) stores in chemoreceptor cells of the rabbit carotid body: significance for chemoreception

Thenotion that intracellular Ca²⁺ (Ca_i²⁺)stores play a significant role in the chemoreception process inchemoreceptor cells of the carotid body (CB) appears in the literaturein a recurrent manner. However, the structural identity of theCa²⁺ stores and their real significance in the function ofchemoreceptor cells are unknown. To assess the functional significanceof Ca_i²⁺ stores in chemoreceptor cells, we havemonitored 1) the release of catecholamines (CA) from thecells using an in vitro preparation of intact rabbit CB and2) the intracellular Ca²⁺ concentration([Ca²⁺]_i) using isolated chemoreceptor cells;both parameters were measured in the absence or the presence of agentsinterfering with the storage of Ca²⁺. We found thatthreshold [Ca²⁺]_i for high extracellularK⁺ (K_e⁺) to elicit a release response is250 nM. Caffeine (10-40 mM), ryanodine (0.5 µM), thapsigargin(0.05-1 µM), and cyclopiazonic acid (10 µM) did not alter thebasal or the stimulus (hypoxia, high K_e⁺)-inducedrelease of CA. The same agents produced Ca_i²⁺transients of amplitude below secretory threshold; ryanodine (0.5 µM), thapsigargin (1 µM), and cyclopiazonic acid (10 µM) did notalter the magnitude or time course of the Ca_i²⁺responses elicited by high K_e⁺. Several potentialactivators of the phospholipase C system (bethanechol, ATP, andbradykinin), and thereby of inositol 1,4,5-trisphosphate receptors,produced minimal or no changes in [Ca²⁺]_i anddid not affect the basal release of CA. It is concluded that, in therabbit CB chemoreceptor cells, Ca_i²⁺ stores do not playa significant role in the instant-to-instant chemoreception process.

     

Cytoplasmic Ca2+ has No Effect on the Excitability of Chara Plasmalemma

Internodal cells of Chara australis were subjected to two consecutiveintracellular perfusions with a Ca²⁺-free EGTA medium whichdisintegrated the tonoplast within about 10 minutes and thenwith a Ca²⁺-buffered medium. All perfusion media usually contained1 mM ATP. To stop the electrogenic pump, the internode was depletedof intracellular ATP. The excitability of the plasmalemma wasnot significantly influenced by intracellular free Ca²⁺ concentrationsup to 10^–4 M. To trigger action potentials, minimum currentdensities of 1 to 2 µA cm^–2 had to be applied atall tested Ca²⁺ concentrations. In the absence of cytoplasmicATP, excitability was completely lost at all Ca²⁺ concentrations. ¹ Present address: Botanisches Institut der Universit?t Bonn,Venusbergweg 22, D-5300 Bonn, FRG. (Received September 22, 1984; Accepted March 6, 1985)     

Regulation of cytoplasmic calcium levels by two nitric oxide receptors

We examined the effects of dissolved nitric oxide (NO) gas oncytoplasmic calcium levels ([Ca²⁺]_i) in C6glioma cells under anoxic conditions. The maximum elevation (27 ± 3 nM) of [Ca²⁺]_i was reached at 10 µM NO. Asecond application of NO was ineffective if the first was >0.5 µM.The NO donor diethylamine/NO mimicked the effects of NO. Acute exposureof the cells to low calcium levels was without effect on the NO-evokedresponse. Thapsigargin (TG) increased [Ca²⁺]_iand was less effective if cells were pretreated with NO. Hemoglobin inhibited the effects of NO at a molar ratio of 10:1. 8-Bromo-cGMP waswithout effect on the NO-evoked response. If cells were pretreated withTG or exposed chronically to nominal amounts of calcium, NO decreased[Ca²⁺]_i. The results suggest that C6 gliomacells have two receptors for NO. One receptor (NO_A)elevates [Ca²⁺]_i and resides on theendoplasmic reticulum (ER). The other receptor (NO_B)decreases [Ca²⁺]_i and resides on theplasmalemma or the ER. The latter receptor dominates when the level ofcalcium within intracellular stores is diminished.

     

Stretch-induced Ca(2+) release via an IP(3)-insensitive Ca(2+) channel

Various mechanicalstimuli increase the intracellular Ca²⁺ concentration([Ca²⁺]_i) in vascular smooth muscle cells(VSMC). A part of the increase in [Ca²⁺]_i isdue to the release of Ca²⁺ from intracellular stores. Wehave investigated the effect of mechanical stimulation produced bycyclical stretch on the release of Ca²⁺ from theintracellular stores. Permeabilized VSMC loaded with ⁴⁵Ca²⁺ were subjected to 7.5% average (15%maximal) cyclical stretch. This resulted in an increase in⁴⁵Ca²⁺ rate constant by 0.126 ± 0.0035. Inhibition of inositol 1,4,5-trisphosphate (IP₃),ryanodine, and nicotinic acid adenine dinucleotide phosphate channels(NAADP) with 50 µg/ml heparin, 50 µM ruthenium red, and 25 µMthio-NADP, respectively, did not block the increase in⁴⁵Ca²⁺ efflux in response to cyclical stretch.However, 10 µM lanthanum, 10 µM gadolinium, and 10 µMcytochalasin D but not 10 µM nocodazole inhibited the increase in⁴⁵Ca²⁺ efflux. This supports the existence of anovel stretch-sensitive intracellular Ca²⁺ store in VSMCthat is distinct from the IP₃-, ryanodine-, and NAADP-sensitive stores.

     

Protective Effect of External Ca2+ on Elongation and the Intracellular Concentration of K+ in Intact Mung Bean Roots under High NaCl Stress

The effects of Ca²⁺ in the external medium on intact mung beanroots under high NaCl stress were investigated. With increasingexternal concentrations of NaCl, mung bean roots showed suppressionof elongation and a decrease in the intracellular concentrationof K⁺. Addition of Ca²⁺ to the external medium alleviated theinhibition of root elongation under the high NaCl stress andmaintained a high intracellular concentration of K⁺ in the elongatingregion of the roots. This counter effect of Ca²⁺ against theNaCl stress on roots was correlated with the ratio of [Ca²⁺]/[Na⁺]²in the external medium. A value above 5.0 ? 10^–4 mM^–1resulted in almost complete recovery of root elongation undervarious high concentrations of NaCl. Root elongation for 24h under NaCl stress was correlated with the extent to whichthe intracellular concentration of K⁺ was in excess of 10 mM.Maintenance of an adequate concentration of K⁺ in root cellsis essential for root elongation under salt stress. These findingsindicate that Ca²⁺ prevents the leakage of intracellular K⁺and thereby supports the elongation of roots under salt stress. (Received November 13, 1989; Accepted June 5, 1990)     

Synthesis of an S-(2-Aminoethyl)-L-Cysteine Conjugate in S-(2-Aminoethyl)-L-Cysteine- Resistant Adenine-Auxotrophic Cells of Datura innoxia Mill

A line of S-(2-aminoethyl)-L-cysteine-resistant adenine-auxotrophiccells (Ad^–AEC^r strain) was isolated from adenine-auxotrophiccells (Ad^– strain) of Datura innoxia Mill by a stepwiseselection method. Ad^–AEC^r and Bl cells, which were clonedfrom the original Ad^–AEC^r cells, were able to grow activelyon medium that contained 10 mM S-(2-aminoethyl)-L-cysteine (AEC),whereas the growth of Ad^– cells ceased completely in thepresence of 0.5 mM AEC. The resistant phenotype has been maintainedfor at least 10 months in culture on medium without AEC. Levels of free lysine in Ad^–AEC^r and Bl cells were similarto that in Ad^– cells. By contrast, the level of free AECin Ad^–AEC cells was 10-fold lower than in Ad^– cellsand no free AEC was detectable in Bl cells. However, acid hydrolysisof extracts from Ad^–AEC^r and Bl cells resulted in a remarkableincrease in levels of detectable AEC. This result indicatesthat conjugated AEC is synthesized and accumulated in the AEC-resistantcells. The level of the AEC conjugate in Bl cells increasedwith increases in the concentration of AEC in the culture medium,while intracellular levels of AEC were so low as not to be detectablein the case of cells grown on medium supplemented with AEC atless than 1 mM. The AEC conjugate was also detected in Ad^–cells, but at lower levels than in the AEC-resistant cells.In addition, AEC was found to be incorporated into soluble proteinsin Ad^– cells. These results suggest that the resistance of AEC-resistant cellsof Datura innoxia is accomplished via acceleration of the synthesisof the AEC conjugate which prevents any increase in intracellularlevels of free AEC. ¹Present address: Institute for Biology and Chemistry, TsumuraCo.Ltd., Inashiki, Ibaraki, 300-03 Japan. ²Present address: North Kanto Shop, Sakata Seed Co. Ltd.,Saitama,347 Japan.     

Intracellular Calcium Increase in Gerbil Taste Cell by Amino Acid Sweeteners

Gustatory transduction mechanisms for sucrose and amino acidsweeteners in gerbil taste cells were studied with Ca²⁺ imagingand whole cell recording techniques. A 100 mM sucrose stimuluswith Ca²⁺ increased the intracellular Ca²⁺ concentration ([Ca²⁺]i)in sweet-sensitive taste cells of the taste bud, but the sucrosestimulus without Ca²⁺ did not change the [Ca²⁺]i. A 10 mM D-phenylalaninesweet stimulus with or without Ca²⁺ similarly increased the[Ca²⁺]i in the taste bud. The addition of 5 µM ionomysin,a Ca²⁺-ionophore, without Ca²⁺ greatly increased the [Ca²⁺]iin the taste bud. The application of 10 mM D-phenylalanine stimuluswithout Ca²⁺ enhanced the outward K⁺ current in isolated tastecells. These results suggest that a sugar sweetener such assucrose induces a depolarization in gerbil taste cells whichactivates voltage-dependent Ca²⁺ channels and that a non-sugarsweetener such as D-phenylalanine releases Ca²⁺ from the internalstores without a depolarization. Chem. Senses 22: 83–91,1997.     

Changes in Outward K+ Currents in Response to Two Types of Sweeteners in Sweet Taste Transduction of Gerbil Taste Cells

Using the whole cell patch clamp technique, we measured changesin outward K⁺ currents of gerbil taste cells in response todifferent kinds of sweeteners. Outward K⁺ currents of the tastecell induced by depolarizing pulses were suppressed by sweetstimuli such as 10 mM Na-saccharin. The membrane-permeable analogof cAMP, cpt-cAMP, also decreased outward K⁺ currents. On theother hand, the K+ currents were enhanced by amino acid sweetenerssuch as 10 mM D-tryptophan. The outward K⁺ current was enhancedby external application of Ca²⁺-transporting ionophore, 5 µMionomycin, and intracellular application of 5 µM inositol-1,4,5-trisphosphate(IP₃). The outward K⁺ currents were no longer suppressed by10 mM Na-saccharin containing 20 µM gurmarin, but werestill enhanced by 10 mM D-tryptophan containing 20 µMgurmarin. These results suggest that sweet taste transductionfor one group of sweeteners such as Na-saccharin in gerbilsis concerned with an increase of the intracellular cAMP level,and that the transduction for the other group of sweetenerssuch as D-tryptophan is concerned with an increase of the intracellularIP₃ level which releases Ca²⁺ from the internal stores. Chem.Senses 22: 163–169, 1997.     

Determination of Volume of Dunaliella Cells by Lithium Dilution Measurements and Derivation of Internal Solute Concentrations

A method has been developed to measure the cell volume of theunicellular green alga Dunaliella parva 19/9 using Li⁺ measurementsonly. Concentrations of internal solutes can also be calculatedif they are assayed in the same samples as Li⁺. We found thatD. parva cells grown in 0.4 kmol m^–3 NaCl have an averageaqueous cell volume of 65.1 ?2.9 µm³, a K⁺ concentrationof 126?6 mol m^–3, a Na⁺ concentration of 11?11 mol m^–3and a glycerol concentration of 615?27 mol m–3 (n= 12).Algae grown in 1.5 kmol m^–3 NaCl have an average aqueouscell volume of 131 ?7.5 µm³, a K⁺ concentration of 109?4mol m^–3, a Na⁺ concentration of 10?39 mol m^–3 anda glycerol concentration of 1 425?59 mol m^–3 (n = 12).These results indicate that D. parva cells adapted to high salinitieshave larger cell volumes than those adapted to lower salinities.However, there is no evidence for a significant difference ininternal Na⁺ concentration, despite the almost 4-fold differencein the concentration of external NaCl. The intracellular glycerolconcentration alone accounts for 65% and 54%, respectively,of the osmotic balance in low and high salt grown cells. Key words: Dunaliella, cell volume, intracellular solutes     

Regulation of phosphate-activated glutaminase (PAG) by glutamate analogues

The ability of structural analogues of glutamate (GLU) to modulate phosphate activated glutaminase (PAG) was assessed in the present series of studies. A number of GLU receptor agonists and antagonists were tested for their ability to inhibit synaptosomal PAG activity. PAG activity was determined by measuring GLU formation from 0.5mM glutamine (GLN) in the presence of 10 mM phosphate. GLU analogues at 5–10 mM were found to significantly inhibit PAG activity. It was determined that PAG inhibition occurred regardless of whether the GLU analogues were receptor agonists or antagonists, however, PAG inhibition was influenced by analogue chain length, isomeric form and substituent substitution. The glutamate uptake blockers, dihydrokainic acid and DL-threo--hydroxyaspartic acid were relatively weak inhibitors of PAG (<25% inhibition) as were the receptor agonists, ibotenic acid and (±)cis-2,3-piperidine-dicarboxylic acid. Other GLU analogues produced inhibition of PAG in the range of 40–70%. PAG inhibition by GLU analogues did not appear to differ substantially among the brain regions evaluated (cortex, striatum and hippocampus). The endogenous amino acids, glycine, taurine and N-acetylaspartic acid, also significantly inhibited PAG activity in the 5–10 mM range. The noncompetitive NMDA antagonists, (+)MK801 and ketamine, at a concentration of 5 mM, significantly stimulated PAG activity 1.5–2 fold over control values. The activation of PAG by (+)MK801 was dose-related, stereoselective and appeared to result from a synergistic interaction with phosphate to enhance substrate (GLN) binding to PAG. The results of these studies suggest that GLU analogues could potentially alter neurotransmitter GLU synthesis if sufficient concentrations of these drugs are used in in vitro or in vivo studies. Furthermore, preliminary evidence suggests that other endogenous amino acids (glycine, taurine, N-acetylaspartic acid) may modulate PAG activity. These studies have further characterized the structural requirements for the allosteric regulation of PAG by glutamate and its analogues.     

Characterization of the Ca2+-Dependent Cl- Efflux in Perfused Chara Cells

The mechanism of the Ca²⁺-dependent Cl^– efflux was studiedin tonoplast-free cells, in which the intracellular chemicalcomposition can be freely controlled. Tonoplast-free cells wereprepared by perfusing the cell interior of internodal cellsof Chara corallina with a medium that contained EGTA. The Ca²⁺-inducedCl^– efflux was measured together with the membrane potentialduring continuous intracellular perfusion. The dependenciesof Cl^– efflux and the membrane potential on the intracellularCa²⁺ or Cl^– concentrations were analyzed. When perfusionwas started with medium that contained Ca²⁺ ions, Cl^–efflux and membrane depolarization were induced. The amountof Cl^– efflux varied considerably among individual cells.The rate of efflux decreased exponentially but a residual effluxremained detectable. The Cl^– efflux was induced at concentrationsof Ca²⁺ ions above 1 µM and reached a maximum at 1 mM.By contrast, the membrane depolarization reached a maximum atabout 10 µM Ca²⁺. The rate of Cl^– efflux increasedlinearly with logarithmic increases in the intracellular Cl^–concentrations. These findings suggest that more than two kindsof Ca²⁺-dependent Cl^– channel might be present in theplasma membrane. Addition of ATP or its removal from the perfusion medium didnot affect the Ca²⁺-dependent Cl^– efflux. Calmodulin antagonistsslightly inhibited the Ca²⁺-dependent Cl^– efflux. ¹Present address: Biological Laboratory, Hitotsubashi University,Naka 2-1, Kunitachi, Tokyo, 186 Japan.     

Heterogeneity of calcium stores and elementary release events in canine pulmonary arterial smooth muscle cells

To examine the natureof inositol 1,4,5-trisphosphate (IP₃)-sensitive andryanodine (Ryn)-sensitive Ca²⁺ stores in isolated caninepulmonary arterial smooth cells (PASMC), agonist-induced changes inglobal intracellular Ca²⁺ concentration([Ca²⁺]_i) were measured using fura2-AM fluorescence. Properties of elementary local Ca²⁺release events were characterized using fluo 3-AM or fluo 4-AM, incombination with confocal laser scanning microscopy. In PASMC, depletion of sarcoplasmic reticulum Ca²⁺ stores with Ryn(300 µM) and caffeine (Caf; 10 mM) eliminated subsequent Caf-inducedintracellular Ca²⁺ transients but had little or no effecton the initial IP₃-mediated intracellular Ca²⁺transient induced by ANG II (1 µM). Cyclopiazonic acid (CPA; 10 µM) abolished IP₃-induced intracellularCa²⁺ transients but failed to attenuate the initialCaf-induced intracellular Ca²⁺ transient. These resultssuggest that in canine PASMC, IP₃-, and Ryn-sensitiveCa²⁺ stores are organized into spatially distinctcompartments while similar experiments in canine renal arterial smoothmuscle cells (RASMC) reveal that these Ca²⁺ stores arespatially conjoined. In PASMC, spontaneous local intracellular Ca²⁺ transients sensitive to modulation by Caf and Ryn weredetected, exhibiting spatial-temporal characteristics similar to thosepreviously described for "Ca²⁺ sparks" in cardiac andother types of smooth muscle cells. After depletion of Ryn-sensitiveCa²⁺ stores, ANG II (8 nM) induced slow, sustained[Ca²⁺]_i increases originating at sites nearthe cell surface, which were abolished by depleting IP₃stores. Discrete quantal-like events expected due to the coordinatedopening of IP₃ receptor clusters ("Ca²⁺puffs") were not observed. These data provide new information regarding the functional properties and organization of intracellular Ca²⁺ stores and elementary Ca²⁺ release eventsin isolated PASMC.

     

A method for maintaining the viability of less hardy plant cells after immersion in liquid nitrogen

Less hardy cells survived immersion in liquid nitrogen by rapidpassage through the growth zone of intracellular ice crystals,provided that cells were partially dehydrated by extracellularfreezing to –10°C or below, after or even withouttreatment with cryoprotective substances. ¹ Contribution No. 1195 from The Institute of Low TemperatureScience. (Received July 6, 1972; )     

Changes in intracellular metabolite pools during growth of adherent MDCK cells in two different media

In bioprocess engineering, the growth of continuous cell lines is mainly studied with respect to the changes in cell concentration, the resulting demand for substrates, and the accumulation of extracellular metabolites. The underlying metabolic process rests upon intracellular metabolite pools and their interaction with enzymes in the form of substrates, products, or allosteric effectors. Here, we quantitatively analyze time courses of 29 intracellular metabolites of adherent Madin–Darby canine kidney cells during cultivation in a serum-containing medium and a serum-free medium. The cells, which originated from the same pre-culture, showed similar overall growth behavior and only slight differences in their demand for the substrates glucose (GLC), glutamine (GLN), and glutamate (GLU). Analysis of intracellular metabolites, which mainly cover the glycolytic pathway, the citric acid cycle, and the nucleotide pools, revealed surprisingly similar dynamics for both cultivation conditions. Instead of a strong influence of the medium, we rather observed a growth phase-specific behavior in glycolysis and in the lower citric acid cycle. Furthermore, analysis of the lower part of glycolysis suggests the well-known regulation of pyruvate kinase by fructose 1,6-bisphosphate. The upper citric acid cycle (citrate, cis-aconitate, and isocitrate) is apparently uncoupled from the lower part (α-ketoglutarate, succinate, fumarate, and malate), which is in line with the characteristics of a truncated cycle. Decreased adenosine triphosphate and guanosine triphosphate pools, as well as a relatively low energy charge soon after inoculation of cells, indicate a high demand for cellular energy and the consumption of nucleotides for biosynthesis. We finally conclude that, with sufficient availability of substrates, the dynamics of GLC and GLN/GLU metabolism is influenced mainly by the cellular growth regime and regulatory function of key enzymes.     

Control of ascorbic acid efflux in rat luteal cells: role of intracellular calcium and oxygen radicals

In luteal cells, prostaglandin (PG)F_2a mobilizes intracellular calcium concentration ([Ca]_i), generates reactive oxygen species (ROS), depletes ascorbic acid (AA) levels, inhibits steroidogenesis, and ultimately induces cell death. We investigated the hypothesis that [Ca]_i mobilization stimulates ROS, which results in depletion of cellular AA in rat luteal cells. We used a self-referencing AA-selective electrode that noninvasively measures AA flux at the extended boundary layer of single cells and fluorescence microscopy with fura 2 and dichlorofluorescein diacetate (DCF-DA) to measure [Ca]_i and ROS, respectively. Menadione, a generator of intracellular superoxide radical (), PGF_2a, and calcium ionophore were shown to increase [Ca]_i and stimulate intracellular ROS. With calcium ionophore and PGF_2a, but not menadione, the generation of ROS was dependent on extracellular calcium influx. In unstimulated cells there was a net efflux of AA of 121.5 ± 20.3 fmol · cm^–1 · s^–1 (mean ± SE, n = 8), but in the absence of extracellular calcium the efflux was significantly reduced (10.3 ± 4.9 fmol · cm^–1 · s^–1; n = 5, P < 0.05). PGF_2a and menadione stimulated AA efflux, but calcium ionophore had no significant effect. These data suggest two AA regulatory mechanisms: Under basal conditions, AA efflux is calcium dependent and may represent recycling and maintenance of an antioxidant AA gradient at the plasma membrane. Under luteolytic hormone and/or oxidative stress, AA efflux is stimulated that is independent of extracellular calcium influx or generation of ROS. Although site-specific mobilization of calcium pools and ROS cannot be ruled out, the release of AA by PGF_2a-stimulated luteal cells may occur through other signaling pathways. luteolysis; apoptosis; self-referencing microelectrode