UPTAKE OF CALCIUM BY SUBCELLULAR FRACTIONS ISOLATED FROM OUABAIN-TREATED CEREBRAL TISSUES

Abstract— Slices of cerebral cortex were incubated in medium containing 0·75 or 2·8 mM ⁴⁵CaCl₂, in the presence or absence of 0·01–0·1 m m -ouabain. Ouabain induced accumulation of calcium by slices to a maximum of 4 μmoles/g of tissue/hr (0·75 m m -CaCl₂ in the medium) and to 8 μmoles/g of tissue/hr (2·8 m m -CaCl₂ in the medium). Accumulation of Ca²⁺ occurred more slowly than loss of K⁺ from the slices and more closely resembled the pattern of Na⁺ uptake.
Mitochondrial fractions isolated from ouabain-treated slices contained significantly more calcium than controls. Inclusion of EDTA in the homogenization medium resulted in decreased amounts of particulate-bound calcium.
The effect of ouabain on accumulation of calcium is discussed with regard to possible relationships to processes of active and passive transport.     

Potassium dependence and phytoplankton ecology: an experimental study

SUMMARY 1. Unialgal cultures of three species common in the freshwater phytoplankton were used to test limitation of specific growth rate and final yield in defined media of low K⁺ concentration (range <0.3–6 μmol L⁻¹ or mmol m⁻³).
2. Growth rate of the diatom Asterionella formosa was independent of K⁺ concentration above 0.7 μmol L⁻¹. Final yield was dependent on initial concentration when accompanied by K⁺ depletion below this concentration, but not by lesser depletion with more residual K⁺. Analyses of particulate K in the biomass indicated a mean final cell content of 2.8 μmol K 10⁻⁸ cells, approximately 1.0% of the organic dry weight.
3. Less detailed work with the diatom Diatoma elongatum showed no dependence of growth rate or final yield upon the initial K⁺ concentration in the range 0.8–3.2 μmol L⁻¹. The phytoflagellate Plagioselmis nannoplanctica suffered net mortality in the lowest concentration tested, 0.8 μmol L⁻¹.
4. Comparison with the range of K⁺ concentration in natural fresh waters, including a depletion induced by an aquatic macrophyte, suggests that K⁺ is unlikely to limit growth of phytoplankton. Nevertheless, there can be correlation of K⁺ with lake trophy.     

TURNOVER OF PROTEIN PHOSPHORUS IN RESPIRING SLICES OF GUINEA PIG CEREBRAL CORTEX: CELLULAR LOCALIZATION OF PHOSPHOPROTEIN SENSITIVE TO ELECTRICAL STIMULATION

Abstract— In experiments designed to localize the increased turnover of phosphoprotein-P which occurs in respiring brain slices as a result of electrical stimulation, a cell separation procedure was used to prepare a fraction enriched in neuronal cell bodies from incubated slices labelled with [³²P]phosphate. Labelled phosphoprotein was found to be twice as concentrated in the neuron-enriched fraction as in other fractions. Electrical stimulation for 10 s increased the rate of incorporation of [³²P]phosphate into phosphoproteins in the neuron-enriched fraction by 25 per cent ( P < 0.05), but had no effect on incorporation into a partially purified glial fraction contaminated with neuropil and cell debris.     

CALCIUM METABOLISM IN ISOLATED BRAIN CELLS AND SUBCELLULAR FRACTIONS

Abstract— The accumulation of calcium ions by brain mitochondria and microsomes and by fractions containing neuronal or glial cells has been studied in vitro with techniques involving ⁴⁵Ca and ultramicro-flame photometry. ATP and substrate-supported calcium accumulation by brain mitochondria was of the same magnitude as for mitochondria from other organs. Brain microsomes accumulated calcium approximately 15 times less than brain mitochondria. Variations in Na⁺/K⁺ ratios and in ATP/ADP ratios had a more marked influence on microsomal uptake than on mitochondrial uptake. The passive Ca²⁺ binding by glial cells was higher than neuronal perikarya and synaptosomes. Also the calcium accumulation ability in cell suspensions was slightly higher for glial cells as compared to neuronal perikarya. The calcium uptake by glial cells was stimulated by high external K⁺ concentration, which also was the case for nerve endings. The uptake in neuronal perikarya was unaffected by variations in K⁺ concentration. A comparison between neuronal and glial mitochondria showed that both reach a steady state level of similar magnitude, but that the rate of initial accumulation was greater for glial mitochondria. A high glial calcium accumulation was also observed for the microsomal fraction.     

Plasmid transformation of Azospirillum brasilense

Abstract Plasmid transformation of the nitrogen-fixing bacterium Azospirillum brasilense is described. A modification of the method of Hanahan [1] was used to transform this bacterium with the 20-kb plasmid pRK290. The efficiency of transformation ranged from 200–1000 transformants per μg of plasmid DNA according to DNA concentration. Ca²⁺, Mn²⁺ and K⁺ were essential for competence, while Rb⁺ and hexamine cobalt(III) chloride did not appear necessary. The length and the temperature of heat-pulse during transformation affected the efficiency of transformation. The response to different numbers of plasmid molecules was linear, in the range of 0.05–1.0 μg of DNA. No transformants were obtained with pRK290 plasmid DNA linearized with Eco RI. The transformability of different strains of Azospirillum has been compared.     

Growth, contents of K+ and kinetics of K+(86Rb) uptake in barley cultured at different low supply rates of potassium

Plants of barley ( Hordeum vulgare L. cv. Salve) were grown with 6.5–35% relative increase of K⁺ supply per day (RKR) using a special computer-controlled culture unit. After a few days on the culture solution the plants adapted their relative growth rate (RGR) to the rate of nutrient supply. The roots of the plants remained in a low salt status irrespective of the rate of nutrient supply, whereas the concentration of K⁺ in shoots increased with RKR. Both V_max and K_m for K⁺(⁸⁶Rb) influx increased with RKR. It is concluded that with a continuous and stable K⁺ stress, the K⁺ uptake system is adjusted to provide an effective K⁺ uptake at each given RKR. Allosteric regulation of K⁺ influx does not occur and efflux of K⁺ is very small.     

EFFECTS OF POTASSIUM AND GLUTAMATE ON BRAIN CORTEX SLICES: UPTAKE AND RELEASE OF GLUTAMIC AND OTHER AMINO ACIDS

Abstract— Effects of an increased concentration of K⁺ (55 m m ) in the medium on fluxes of glutamate and other amino acids in the presence and absence of 10 m m -glutamate were studied. The following observations were made:
(1) The efflux of glutamate is slightly increased by excess K⁺. The glutamate efflux is smaller than the potassium fluxes.
(2) The K⁺-induced increase of glutamate efflux is enhanced under anoxia or in glutamate-containing media.
(3) The influx of glutamate is unaffected or slightly increased by excess K⁺.
(4) The efflux of GABA is increased by excess K⁺, both in the absence and in the presence of glutamate.
(5) Efflux of glutamine, leucine and lysine is increased by excess K⁺, but only provided that glutamate is also present in the medium.
(6) Efflux of glutamate and of GABA is increased by addition of 10 m m -glutamate.     

Probable Role of Allylisothiocyanate-Sensitive H+, K+-ATPase in Spicule Calcification in Embryos of the Sea Urchin, Hemicentrotus pulcherrimus

In embryos of the sea urchin, Hemicentrotus pulcherrimus , as well as in cultured cells derived from isolated micromeres, spicule formation was inhibited by allylisothiocyanate, an inhibitor of H⁺, K⁺-ATPase, at above 0.5 μM and was almost completely blocked at above 10 μM. Amiloride, an inhibitor of Na⁺, H⁺ antiporter, at above 100 μM exerted only slight inhibitory effect, if any, on spicule formation. Intravesicular acidification, determined using [ dimethylamine -¹⁴C]-aminopyrine as a pH probe, was observed in the presence of ATP and 200 mM KCl in microsome fraction obtained from embryos at the post gastrula stage, at which embryos underwent spicule calcification. Intravesicular acidification and K⁺-dependent ATPase activity were almost completely inhibited by allylisothiocyanate at 10 μM. Allylisothiocyanate-sensitive ATPase activity was found mainly in the mesenchyme cells with spicules isolated from prisms. H⁺, K⁺-ATPase, an H⁺ pump, probably mediates H⁺ release to accelerate CaCO₃ deposition from Ca²⁺, CO₂ and H₂O in the primary mesenchyme cells. Intravesicular acidification was stimulated by valinomycin at the late gastrula and the prism stages but not at the pluteus stage. K⁺ permeability probably increases after the prism stage to activate H⁺ release.     

The use of a perfused, whole-body preparation to measure the branchial and intestinal influx of 137-caesium in the rainbow trout (Oncorhynchus mykiss)

The branchial and intestinal influx of caesium (Cs) in the rainbow trout ( Oncorhynchus mykiss ) were measured using a perfused whole-body preparation. The branchial influx of Cs was small, 0–31 μmoles kg⁻¹ h⁻¹ at an external concentration of 1 mm. Branchial Cs influx was saturable, with a K_m of 1–92 mm and a J_max of l.05μmoles kg⁻¹ h⁻¹. Intestinal Cs influx was not saturable, but was directly proportional to the mucosal Cs concentration. Intestinal Cs influx was approximately 10–40 times greater than branchial Cs influx over a wide range of external Cs concentrations. These results are discussed with respect to mechanisms of Cs uptake and to the relative accumulation of radiocaesium from water and food in the environment.     

Hydrogen peroxide formation in cultured rose cells in response to UV-C radiation

Suspension-cultured rose ( Rosa damascena Mill. cv. Gloire de Guilan) cells irradiated with UV-C (254 nm. 558 J m⁻²) showed a transient production of H₂O₂ as measured by chemiluminescence of luminol in the presence of peroxidase (EC 1.1 1.1.7). The peak concentration of H₂O₂, which occurred at about 60–90 min after irradiation, was 8–9 μ M . The time course for the appearance of H₂O₂ matched that for UV–induced K⁺ efflux. Treatments that inhibited the UV-induced efflux of K⁺, including heat and overnight incubation with cycloheximide and diethylmaleate, also inhibited the appearance of H₂O₂. The converse was not always true, since catalase (EC 1.11.1.6. and salicylhydroxamic acid, which inhibited luminescence, did not stop K⁺ efflux. We conclude that H₂O₂ synthesis depends on K⁺ efflux. Because H₂.O₂ in the extracellular space is required for lignin synthesis in many plant tissues, we suggest that the UV–stimulated production of H₂O₂ is an integral part of a defensive lignin synthesis.     

RETENTION AND EXCHANGE OF POTASSIUM IN A SYNAPTOSOMAL FRACTION OF MOUSE BRAIN

Abstract— Myelin, synaptosomal and mitochondrial fractions obtained from homogenates of whole mouse brain contain K⁺ which can exchange with ⁴²K⁺ at 2º in 0·32 m -sucrose. The content and rates of exchange of K⁺ were greater at pH 8·2 than at 6·1. In the synaptosomal preparations, the rates of exchange and content of ⁴²K⁺ and K⁺ declined progressively with decreasing pH.
Of the total synaptosomal K⁺, 95 per cent could exchange with external ⁴²K⁺. At pH 7·5, 20 per cent of the K⁺ and 78 per cent of the Na⁺ appeared to reside in osmotically insensitive pools. Synaptosomal K⁺ at 2º was slowly displaced by NaCl (0·18 m ) and the rate of exchange between ⁴²K⁺ and K⁺ was retarded. KCI (0·18 m ) did not readily displace endogenous Na⁺. Synaptosomal K⁺ exchanged with exogenous K⁺ more rapidly than with exogenous Na⁺.
These observations have been discussed in terms of possible roles for ion exchange as the principal means by which K⁺ traverses the plasma membrane at 2º.     

Basic and Regulatory Mechanisms of In Vitro Release of Met-Enkephalin from the Dorsal Zone of the Rat Spinal Cord

Abstract: Under control conditions, superfused slices of the dorsal half of the lumbar enlargement from adult rats released Met-enkephalin-like material (MELM) that behaved as authentic Met-enkephalin under two different chromatographic procedures (Bio-gel filtration, HPLC). MELM release increased markedly on exposure of slices to batrachotoxin (0.5 μ M ) or to an excess of K⁺ (28 and 56 m M instead of 5.6 m M ). The K ⁺ -evoked release was totally dependent on the presence of Ca²⁺ in the super-fusing fluid whereas the spontaneous efflux of MELM was only partially Ca²⁺-dependent. Further experiments performed with tissues of polyarthritic rats indicated that the increase in their MELM levels was associated with a lower fractional rate constant of MELM release, therefore suggesting that spinal Met-enkephalin turnover might be reduced in chronically suffering animals. Examination of the possible modulation of MELM release by various neuroactive compounds present within the dorsal horn revealed that cholecystokinin (10 μ M ), but not its desulphated derivative, substance P-sulphoxide (10 μ M ), and to a lesser extent substance P, enhanced the K⁺-evoked MELM release. In contrast, γ-aminobutyric acid (10 μ M ) and (–)-baclofen (1 μ M ) partially prevented the stimulatory effect of K⁺ on MELM release. Other compounds such as serotonin, somatostatin, and neurotensin altered neither the spontaneous nor the K⁺-evoked release of MELM.     

Ethanol Induces Apoptosis in Cerebellar Granule Neurons by Inhibiting Insulin-Like Growth Factor 1 Signaling

Abstract: The ability of ethanol to interfere with insulin-like growth factor 1 (IGF-1)-mediated cell survival was examined in primary cultured cerebellar granule neurons. Cells underwent apoptosis when switched from medium containing 25 m M K⁺ to one containing 5 m M K⁺. IGF-1 protected granule neurons from apoptosis in medium containing 5 m M K⁺. Ethanol inhibited IGF-1-mediated neuronal survival but did not inhibit IGF-1 receptor binding or the neurotrophic action of elevated K⁺, and failed to potentiate cell death in the presence of 5 m M K⁺. Inhibition of neuronal survival by ethanol was not reversed by increasing the concentration of IGF-1. Significant inhibition by ethanol (15–20%) was observed at 1 m M and was half-maximal at 45 m M . The inhibition of IGF-1 protection by ethanol corresponded to a marked reduction in the phosphorylation of insulin receptor substrate 1, the binding of phosphatidylinositol 3-kinase (PI 3-kinase), and a block of IGF-1-stimulated PI 3-kinase activity. The neurotrophic response of IGF-1 was also inhibited by the PI 3-kinase inhibitor LY294002, the protein kinase C inhibitor chelerythrine chloride, and the protein kinase A inhibitor KT5720, but unaffected by the mitogen-activated protein kinase kinase inhibitor PD 98059. These data demonstrate that ethanol promotes cell death in cerebellar granule neurons by inhibiting the antiapoptotic action of IGF-1.     

Opiates Inhibit Acetylcholine Release from Torpedo Nerve Terminals by Blocking Ca2+ Influx

Abstract: In the present communication we report that Ca²⁺-dependent acetylcholine release from K⁺-depolarized Torpedo electric organ synaptosomes is inhibited by morphine, and that this effect is blocked by the opiate antagonist naloxone. This finding suggests that the purely cholinergic Torpedo electric organ neurons contain pre-synaptic opiate receptors whose activation inhibits acetylcholine release. The mechanisms underlying this opiate inhibition were investigated by comparing the effects of morphine on acetylcholine release induced by K⁺ depolarization and by the Ca²⁺ ionophore A23187 and by examining the effect of morphine on ⁴⁵Ca²⁺ influx into Torpedo nerve terminals. These experiments revealed that morphine inhibits ⁴⁵Ca²⁺ influx into K⁺-depolarized Torpedo synaptosomes and that this effect is blocked by naloxone. The effects of morphine on K⁺ depolarization-mediated ⁴⁵Ca²⁺ influx and on acetylcholine release have similar dose dependencies (half-maximal inhibition at 0.5–1 μ M ), suggesting that opiate inhibition of release is due to blockage of the presynaptic voltage-dependent Ca²⁺ channel. This conclusion is supported by the finding that morphine does not inhibit acetylcholine release when the Ca²⁺ channel is bypassed by introducing Ca²⁺ into the Torpedo nerve terminals via the Ca²⁺ ionophore.     

Cell death in an ischemic infarct rim model

Using an in vitro model that simulates the microenvironment in the ischemic infarct rim, we have examined the temporal profile and possible mechanisms of cell death in the neuropil (an astrocyte-rich area or ARA) of organotypic hippocampal slice cultures. Two-photon confocal microscopy, propidium iodide, and GFAP-GFP transgenic mice were used to confirm cell death in astrocytes. An 'ischemic solution' (IS) induced major cell death throughout the hippocampus over 24 h, with the earliest injury starting in ARA. Our studies using IS or ion replacements in IS revealed that cell death in ARA was modest when K⁺ was increased or pH lowered. High K⁺ is most effective in reducing cell death when HCO₃⁻ is normal or high. When Cl⁻ or HCO₃⁻ was reduced, cell injury was worsened. 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) protected cells from IS-induced death in a dose-dependent manner (1–4000 μmol/L). We conclude that (i) various areas of the hippocampal formation respond differently to ionic replacements; (ii) K⁺ interacts with other ions to protect cells in ARA; and (iii) DIDS has a substantial protective effect in ARA by blocking DIDS-sensitive membrane exchangers or by interfering with intracellular signaling pathways.     

Potassium ion channels in the plasmalemma

The potassium ion is an indispensible cytosolic component of living cells and a key osmolyte of plant cells, crossing the plasmalemma to drive physiological processes like cell growth and motor cell activity. K⁺ transport across the plasmalemma may be passive through channels, driven by the electrochemical gradient, K⁺ equilibrium potential (E_K) – membrane potential (V_m), or secondary active by coupling through a carrier to the inward driving force of H⁺ or Na⁺. Known K⁺ channels are permeable to monovalent cations, a permeability order being K⁺ > Rb⁺ > NH₄⁺ > Na⁺≥ Li⁺ > Cs⁺. The macroscopic K⁺ currents across a cell or protoplast surface commonly show rectification, i.e. a V^m-dependent conductance which in turn, may be controlled by the cytosolic activity of Ca²⁺, of K⁺, of H⁺, or by the K⁺ driving force. Analysis by the patch clamp technique reveals that plant K⁺ channels are similar to animal channels in their single channel conductance (4 to 100 pS), but different in that a given channel population slowly activates and may not inactivate at all. Single-channel kinetics reveal a broad range of open times (ms to s) and closed times (up to 100 s). Further progress in elucidating plant K⁺ channels will critically depend on molecular cloning, and the availability of channel-specific (phyto)toxins.     

Long-term effects of abscisic acid on K+ transport in young wheat plants of different K+ status

The effects of abscisic acid (ABA) on growth, uptake and translocation of potassium ions, K⁺,Mg²⁺-ATPase activity and transpiration were investigated in young wheat ( Triticum aestivum L. cv. Martonvásári-8) plants grown at different K⁺ supplies. Long-term treatment with ABA (10 μ M ) reduced growth in high-K⁺ plants, but had less effect under low-K⁺ conditions. K⁺(⁸⁶Rb) uptake was inhibited by about 70 and 40% in low- and high-K⁺ plants, respectively. The stimulation by K⁺ of the Mg²⁺-ATPase activity in the root microsomal fraction was lost with ABA treatment. It is suggested that the inhibitory effect of ABA on K⁺ uptake may be related to this effects on the K⁺,Mg²⁺-ATPase. Translocation of K⁺ to the shoot was inhibited in low-K⁺ plants only, and it was not affected in high-K⁺ plants. In parallel to this, ABA treatment reduced transpiration by about 50% in low-K⁺ plants, whereas a much smaller effect was seen in high-K⁺ plants. These observations suggest that the regulation by ABA of the stomatal movements is strongly counteracted by high-K⁺ status.     

Molecular determinants of the Arabidopsis AKT1 K+ channel ionic selectivity investigated by expression in yeast of randomly mutated channels

The Arabidopsis thaliana K⁺ channel AKT1 was expressed in a yeast strain defective for K⁺ uptake at low K⁺ concentrations (<3 m M ). Besides restoring K⁺ transport in this strain, AKT1 expression increased its tolerance to salt (NaCl or LiCl), whatever the external K⁺ concentration used (50 μ M , 5 m M , or 50 m M ). We took advantage of the latter phenomenon for screening a library of channels randomly mutated in the region that shares homologies with the pore forming domain (the so-called P domain) of animal K⁺ channels (Shaker family). Cassette mutagenesis was performed using a degenerate oligonucleotide that was designed to ensure, theoretically, a single mutation per P cassette. The mean number of amino acid exchanges per cassette turned out to be 1.4. Mutant channels that conferred on the transformed cells a reduction in salt tolerance (increased Na⁺ content, decreased K⁺ content, and lower growth rate, as compared to control cells expressing the wild-type channel) were selected. By co-expressing them with the wild-type AKT1 cDNA, it was shown that the mutated polypeptides were expressed, stable and correctly targeted to the cell membrane where they formed channels with altered properties. Analysis of the mutation distribution in these channels suggests that the AKT1 P domain has a structure similar to that of animal Shaker channels (a strongly constrained central region lining the tunnel that includes the highly conserved consensus motif TXXTXGYGD, and flanking regions forming the outer mouth of the pore), with an additional selectivity filter located upstream from the tunnel and formed by residues present in the N-terminal flanking region.     

Rubidium Uptake and Accumulation in Peripheral Myelinated Internodal Axons and Schwann Cells

Abstract: To study mechanisms of K⁺ transport in peripheral nerve, uptake of rubidium (Rb⁺), a K⁺ tracer, was characterized in rat tibial nerve myelinated axons and glia. Isolated nerve segments were perfused with zero-K⁺ Ringer's solutions containing Rb⁺ (1–20 m M ) and x-ray microanalysis was used to measure water content and concentrations of Rb, Na, K, and Cl in internodal axoplasm, mitochondria, and Schwann cell cytoplasm and myelin. Both axons and Schwann cells were capable of removing extracellular Rb⁺ (Rb⁺_o) and exchanging it for internal K⁺. Uptake into axoplasm, Schwann cytoplasm, and myelin was a saturable process over the 1–10 m M Rb⁺_o concentration range, although corresponding axoplasmic uptake rates were higher than respective glial velocities. Mitochondrial accumulation was a linear function of axoplasmic Rb⁺ concentrations, which suggests involvement of a nonenzymatic process. At 20 m M Rb⁺_o, a differential stimulatory response was observed; i.e., axoplasmic Rb⁺ uptake velocities increased more than fivefold relative to the 10 m M rate, and glial cytoplasmic uptake rose almost threefold. Finally, Rb⁺_o uptake rate into axons and glia was completely inhibited by ouabain (2–4 m M ) exposure or incubation at 4°C. These results suggest that Rb⁺ uptake into peripheral nerve internodal axons and Schwann cells is mediated by Na⁺,K⁺-ATPase activity and implicate the presence of axonal- and glial-specific Na⁺ pump isozymes.     

Effects of nitrate on influx, efflux and translocation of potassium in young sunflower plants

Influx, efflux and translocation of K⁺(⁸⁶Rb) were studied in the roots of sunflower seedlings ( Helianthus annuus L. cv. Uniflorus) treated with 0–4.0 m M NO₃⁻ during a 9 day growth period or a 24 h pretreatment period. Roots treated with high levels of NO₃⁻ absorbed and translocated more K⁺(⁸⁶Rb) than seedlings treated with low levels of NO₃⁻. The content of K⁺ in the shoots was, however, higher in seedlings treated with low levels of NO₃⁻, indicating a low rate of retranslocation of K⁺ in those plants. K⁺(⁸⁶Rb) efflux was highest into the low-NO₃⁻ solutions. All effects on K⁺(⁸⁶Rb)-fluxes were more obvious in high-K plants than in low-K plants. The results are discussed in relation to the Dijkshoorn-Ben Zioni hypothesis for K⁺+ NO₃⁻-uptake and translocation in plants.