Release of [3H]serotonin from brain slices

1. Labelled serotonin ([³H]5-HT) accumulated by slices of rat brain either in vivo or in vitro is released by depolarizing procedures such as electrical stimulation or high external potassium concentrations. Electrical stimulation predominantly affects the liberation of the unchanged amine, rather than of its principal metabolite, 5-HIAA. 2. Release of [³H]5-HT does not appear to be calcium-dependent. 3. Amount of release parallels the density of serotonin-containing nerve terminals in each of several cerebral regions tested. Release from several extracerebral tissues was similar to that obtained from cerebral tissues having relatively little endogenous 5-HT. 4. Electrically induced release of [³H]5-HT is markedly inhibited by desipramine, chlorpromazine, LSD, lithium and ouabain.     

Calcium- and voltage-dependence of nematocyst discharge in Hydra vulgaris

In Hydra vulgaris, discharge of stenotele nematocysts was induced by contact with prey, electrical stimuli, or increase in the external potassium concentration. In each case 10^-4 M calcium was required in the culture medium. The results indicated a voltage- and calcium-dependent mechanism different from mechano- or chemoreception allowing calcium influx from the external medium. A threshold for activation was suggested by the steep increase of the rate of electrically induced discharge in external fields of 3.5 kV/m. Although organic antagonists for vertebrate calcium channels were ineffective in blocking the calcium-induced nematocyst discharge, inorganic divalent and trivalent cations competitively inhibited the process, with a sequence (Co²⁺ < Ni²⁺ < Cd²⁺ < La³⁺ < Gd³⁺) similar to that seen for antagonism of calcium influx through voltage-dependent channels. Magnesium, an intracellular calcium antagonist, decreased nematocyst discharge, while strontium replacing calcium supported the discharge at a lowered rate. It is concluded that in the nematocyte a voltage-activated influx of calcium through apical ion channels initiates the discharge of the nematocyst in an exocytotic process.     

Effect of external sodium and calcium on calcium efflux in frog striated muscle

Summary The effect of media with different ionic composition on calcium efflux from the dorsal head of semitendinosus muscles ofRana pipiens was studied. The reduction in the fractional loss of⁴⁵Ca, when going from normal Ringer's solution to an ONa–OCa medium, was 60%. Withdrawal of only Na or Ca from the external medium also caused a significant drop in the fractional loss (33% and 34%, respectively). The effect of different concentrations of Ca (studied in the absence of the external Na) was also studied. It was found that a linear function could describe the relationship between the calcium-dependent calcium efflux and the external calcium concentration. These results indicate that calcium efflux from frog muscle fibers consists of three major components: one that is dependent on the presence of calcium in the external medium, one that is dependent on the presence of sodium in the external medium, and one that persists in the absence of these two cations.     

Ca2+-dependent 4-aminopyridine- and α-latrotoxin-evoked [14C] GABA release has different sensitivity to protein kinase inhibitors

Synaptosomes from the rat brain were used to study the influence of protein kinase inhibitors on the plasma membrane calcium permeability and calcium-dependent α-latrotoxin (LTX)-induced [¹⁴C] GABA release. It was shown that stimulated by depolarization with 4-aminopyridine (4-AP) calcium permeability was inhibited by sphingosine. Calcium permeability evoked by LTX was insensitive to either of following agents; sphingosine, staurosporine, and W-7. Unlike pronounced inhibitory effects of sphingosine, staurosporine, or W-7 on 4-AP-evoked [¹⁵C] GABA release, a calcium-dependent secretagogue effect of LTX was not changed by these agents. The mechanism of clacium-dependent LTX-evoked secretagogue effect is discussed.     

NONLINEAR PROCESSES OF INTRACELLULAR CALCIUM SIGNALING AS A TARGET FOR THE INFLUENCE OF EXTREMELY LOW-FREQUENCY FIELDS

Theoretical analysis of peculiarities of reception of weak extremely low-frequency periodic signals by calcium-dependent intracellular regulatory systems was performed on the reduced “minimal” model for calcium oscillations suggested by Goldbeter et al. (Proc. Natl. Acad. Sci. USA 87, 1461–1465, 1990). The model considered the following calcium-dependent processes: the rise in intracellular free calcium concentration ([Ca²⁺]_i) due to calcium ionophore A23187 action on a cell, activation of the Ca²⁺ entry through calcium channels in the plasma membrane by the initial rise in [Ca²⁺]_i, and the Ca²⁺ release from intracellular stores by the calcium-induced calcium release mechanism. Calcium channels of plasma membrane were chosen as a target for the modulating signal and an additive noise influence in the model. An increase in [Ca²⁺]_i under the influence of the modulating signal was demonstrated to depend not only on the amplitude and frequency of this signal, but also on the phase of the signal with respect to a momentary chemical stimulation of the cell. Such an effect was found only at high strengths of chemical stimulation and with a particular sequence of delivery of the chemical and electromagnetic stimuli. An increase in noise intensity led to magnification of the mean level of [Ca²⁺]_i in a narrow frequency range by the mechanism of stochastic resonance. Under the influence of a modulating periodic signal, the gradual increase in strength of chemical stimulation induced a system transition from regular to chaotic behavior, and then to induced periodic oscillations. A boundary of the transition from chaotic to periodic oscillations corresponded to a “threshold” of sensitivity of calcium-dependent intracellular signaling systems on [Ca²⁺]_i to the influence of the modulating signal. Results of the theoretical analysis led us to conclude that the narrow-band response of a system to an external electromagnetic signal is determined purely by nonlinear properties of the system.     

Calcium-regulated phosphorylation of proteins in the membrane-matrix compartment of the Chlamydomonas flagellum

Crosslinking of surface-exposed domains on certain Chlamydomonas flagellar membrane glycoproteins induces their movement within the plane of the flagellar membrane. Previous work has shown that these membrane glycoprotein movements are dependent on a critical concentration of free calcium in the medium and are inhibited reversibly by calcium channel blockers and the protein kinase inhibitors H-7, H-8, and staurosporine. These observations suggest that the flagellum may use a signaling pathway that involves calcium-activated protein phosphorylation to initiate flagellar membrane glycoprotein movements. In order to pursue this hypothesis, we examined the calcium dependence of phosphorylation of flagellar membrane-matrix proteins using an in vitro system containing [γ-³²P]ATP or [³⁵S]ATPγS. Using only endogenous enzymes and endogenous substrates found in the membrane-matrix fraction obtained by extraction of flagella with 0.05% Nonidet P-40, we observed both calcium-independent protein phosphorylation and calcium-dependent protein phosphorylation in addition to an active protein dephosphorylation activity. Addition of micromolar free calcium increased the amount of protein phosphorylation severalfold. Calcium-activated protein kinase activity was inhibited by H-7, H-8, and staurosporine, the same protein kinase inhibitors that inhibit the calcium-dependent glycoprotein redistribution in vivo. A small group of polypeptides in the 26–58 kDa range exhibited a dramatic increase in phosphorylation in the presence of 20 μM free calcium. We suggest that Chlamydomonas utilizes the intraflagellar free calcium concentration to regulate the phosphorylation of specific flagellar proteins in the membrane-matrix fraction, one or more of which may be involved in regulating the machinery responsible for flagellar membrane glycoprotein redistribution.     

Electrophysiology of phagocytic membranes: III. Evidence for a calcium-dependent potassium permeability change during slow hyperpolarizations of activated macrophages

The roles of potassium and calcium in the slow hyperpolarizations of membranes of activated macrophages are investigated using standard intracellular electrical recording techniques.The amplitude of spontaneous slow hyperpolarizations decreases as a logarithmic function of the external potassium concentration in the culture medium. Similar dependence on the potassium gradient is observed when different levels of membrane potentials are imposed by constant current injection. The reversal potential for electrically evoked slow hyperpolarizations is ?90 mV. A 10-fold increase in external potassium concentration causes a 60 mV shift of the reversal potential towards zero.Divalent cation ionophores (A23187 and X537A) can induce slow hyperpolarization responses in quiescent cells or permanent hyperpolarization in spontaneously active cells. The amplitude of the ionophore-induced hyperpolarizations is reduced by an increase in external potassium concentration in a manner consistent with data on slow hyperpolarization responses in the absence of ionophore.The calcium antagonist, verapamil, depresses the slow hyperpolarization responses at the concentration of 10^?5 M.It is suggested that the development of the hyperpolarizing response is due to a calcium-dependent potassium channel. The data support the assumption that spontaneous and artificially elicited slow hyperpolarization responses share a common calcium-dependent mechanism.     

Uptake and release ofmeta-tyramine,para-tyramine,and dopamine in rat striatal slices

The uptakes of high-affinity concentrations (10^–8 M) ofmeta-tyramine (m-TA),para-tyramine (p-TA), and dopamine (DA) into rat striatal slices have been shown to be inhibited by DNP and ouabain. We now demonstrate that cocaine (5×10^–6 M) and low concentrations of sodium ion (26×10^–3 M) also reduced these uptakes. The spontaneous efflux and the release [induced by an elevated concentration of potassium ion (5×10^–2 M)] of each of the previously accumulated amines were studied in the presence and absence of added calcium ions. The spontaneous efflux of each amine (especially the tyramines) was enhanced by the absence of calcium ions. Part of this enhancement seemed to be due to an inhibition of a calcium-dependent reuptake. The elevated concentration of potassium ion proved to be an effective releaser of each amine; and for DA, such release was decreased by the removal of calcium. Form- andp-TA, however, the removal of calcium either did not reduce or completely abolished the releases depending upon the duration of the calcium removal. The significance of these findings is discussed.     

Tryptophan Availability Modulates Serotonin Release from Rat Hypothalamic Slices

Application of a novel in vitro experimental system has allowed us to describe the relationship between tryptophan availability and serotonin release from rat hypothalamic slices. Superfusing hypothalamic slices with a physiologic medium containing l-tryptophan (1, 2, 5, or 10 microM) caused dose-dependent elevations in tissue tryptophan levels; the magnitude of the elevations produced by supplementing the medium with less than 5 microM tryptophan was within the physiologic range for rat brain tryptophan levels. Slice serotonin levels rose biphasically as the tryptophan concentration in the medium was increased. Superfusing the slices with medium supplemented with a low tryptophan concentration (1 or 2 microM) caused proportionally greater incremental changes in serotonin levels than the increases caused by further elevating the tryptophan concentration (5 or 10 microM). The spontaneous release of serotonin from the slices exhibited a dose-dependent relationship with the tryptophan concentration of the superfusion medium. Electrically evoked serotonin release, which was calcium-dependent and tetrodotoxin-sensitive, also increased in proportion to the medium tryptophan concentration. These data suggest that the rate at which serotonin is released from hypothalamic nerve terminals is coupled to brain tryptophan levels. Accelerations in hypothalamic serotonin synthesis, caused by elevating brain tryptophan levels, result in proportionate increases in the rates of serotonin release during rest and with membrane depolarization.     

Spontaneous induction of superoxide release and degranulation of neutrophils in isotonic potassium medium: the role of intracellular calcium

When guinea pig peritoneal neutrophils were suspended in the isotonic medium of potassium, rubidium, and cesium ions at 37 degrees C, the cells released superoxide, while low activity was observed in the isotonic medium of sodium and lithium ions. The activity induced in the potassium medium was enhanced by potassium-ionophores, valinomycin, and gramicidin, and decreased by a potassium channel blocker, 4-aminopyridine. The superoxide-releasing activity was not affected by the presence or absence of extracellular calcium but was inhibited by an intracellular calcium antagonist-8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate(TMB-8) with the half-inhibition concentration of 50 microM. The release of granular enzymes, lysozyme and beta-glucuronidase, was also induced in the isotonic potassium medium in the absence of extracellular calcium and inhibited by TMB-8. A remarkable elevation of the intracellular free calcium concentration in neutrophils, which was monitored by quin-2 fluorescence, was found when the cells were added to the potassium medium without calcium. The elevation was inhibited by the addition of TMB-8. These observations suggest that calcium mobilization from intracellular storage sites, not an influx of calcium from the extracellular medium, causes the release of superoxide and the granular enzymes in isotonic potassium medium.     

Isolation of a New Exopolygalacturonase Producing Digalacturonic Acid from Pectic Acid

Transglutaminases catalyze the cross-linking and amine incorporation of proteins, and are implicated in various biological phenomena such as blood clotting, wound healing, apoptosis, and cell differentiation. Streptomyces lavendulae Y-200, isolated from soil, produced a substance that inhibited transglutaminases. The inhibitory substance was purified from the cultured medium by procedures of acid precipitation, deoxyribonuclease treatment, and gel filtration chromatography. The partially purified sample was dark brown. The inhibitory activity was stable under acidic, alkaline, and high temperature conditions, and resistant to the treatment with proteinases such as trypsin and Pronase. The molecular weight of the inhibitory substance was estimated to be between 10⁴ and 10⁵ from its permeability through ultrafilter membranes. The acid hydrolysate of the inhibitory substance contained amino acids and sugars. The inhibitory substance inhibited both calcium-dependent and calcium-independent transglutaminases in a competitive manner with a glutamine substrate. The extent of inhibition caused by the calcium-dependent transglutaminase increased with increasing calcium concentration. The results obtained here may help identify a novel regulatory substance of transglutaminase in biological systems.     

Effects of systematically administered lithium on tryptophan transport and exchange in plasma-membrane vesicles isolated from rat brain

The effect of lithium on the sodium-dependent high-affinity system for tryptophan uptake was examined in plasma membrane vesicles derived from rat brain. We demonstrated that Na⁺ could be replaced by lithium in the external medium and the presence of lithium produced an increase in theV _max of the tryptophan transport whereas it had no significant effect on theK _m for the substrate. Plasma membrane vesicles derived from synaptosomes obtained from long-term lithium-treated rats are able to accumulate tryptophan to a greater extent than normal rats and maintain a more negative membrane potential than controls. Our data support the idea that the stimulation by lithium of the high-affinity uptake system for tryptophan by maintaining adequate membrane potentials across the membrane, could lead to the stabilization of serotonin production, as has been demonstrated in long termlithium treatment.     

A new method to detect cadmium uptake in protoplasts

The mechanism for cadmium (Cd²⁺) uptake into the cytosol of protoplasts from 5- to 7-day-old wheat seedlings (Triticum aestivum L. cv. Kadett) was investigated by a new method, using fluorescence microscopy and the heavy metal-specific fluorescent dye, 5-nitrobenzothiazole coumarin, BTC-5N. Cadmium fluorescence gradually increased in the cytosol of shoot and root protoplasts upon repeated additions of CdCl₂ to the external medium, reflecting an uptake of Cd²⁺. The uptake was inhibited by calcium and potassium chloride, as well as by Verapamil and tetraethylammonium (TEA), inhibitors of calcium and potassium channels, respectively. Calcium competitively inhibited the cadmium uptake. The metabolic inhibitors vanadate and dinitrophenol partly inhibited the uptake, suggesting it was dependent on membrane potential. The results indicate that cadmium is taken up by channels permeable to both calcium and potassium. The total uptake of cadmium into the protoplasts was also detected by unidirectional flux analyses using ¹⁰⁹Cd²⁺, and showed approximately the same maximal concentration of Cd²⁺ as the fluorescence measurements. By combining the two methods it is possible to detect both uptake into the cytosol and into the vacuole.Abbreviations BTC-5N, AM Acetoxymethyl ester of 5-nitrobenzothiazole coumarin - DNP 2,4-Dinitrophenol - TEA Tetraethylammonium     

Botulinum toxin a inhibits acetylcholine release from cultured neurons in vitro

Summary Clostridium botulinum type toxin A (BoTx) blocks stimulus-induced acetylcholine (ACh) release from presynaptic nerve terminals at peripheral neuromuscular junctions. However, the detailed mechanism of this effect remains elusive. One obstacle in solving this problem is the lack of a suitable in vitro homogenous cholinergic neuronal model system. We studied the clonal pheochromocytoma PC12 cell line to establish such a model. PC12 cells were differentiated in culture by treatment with 50 ng/ml nerve growth factor (NGF) for 4 days to enhance cellular ACh synthesis and release properties. Stimulation of these cells with high K⁺ (80 mM) in the perfusion medium markedly increased calcium-dependent [³H]ACh release compared to undifferentiated cells. Stimulated [³H]ACh release was totally inhibited by pretreatment of cells with 2 nM BoTx for 2 h. BoTx inhibition of [³H]ACh release was time- and concentration-dependent. A 50% inhibition was obtained after 2 h incubation with a low (0.02 nM) toxin concentration. The time required for 2 nM BoTx to cause a measurable inhibition (18%) of stimulated [³H]ACh release was 30 min. Botulinum toxin inhibition of stimulated ACh release was prevented by toxin antiserum and heat treatment, suggesting the specificity of the toxin effect. Our results show that by differentiation with NGF, PC12 cells can be shifted from an insensitive to a sensitive state with respect to BoTx inhibition of stimulated ACh release. This cell line, therefore, may serve as a valuable in vitro cholinergic model system to study the mechanism of action of BoTx.     

The Effect of Spider Toxin PhTx3-4, ω-Conotoxins MVIIA and MVIIC on Glutamate Uptake and on Capsaicin-Induced Glutamate Release and [Ca<Superscript>2+</Superscript>]<Subscript>i</Subscript> in Spinal cord Synaptosomes

In spinal cord synaptosomes, the spider toxin PhTx3-4 inhibited capsaicin-stimulated release of glutamate in both calcium-dependent and -independent manners. In contrast, the conus toxins, ω-conotoxin MVIIA and ω-conotoxin MVIIC, only inhibited calcium-dependent glutamate release. PhTx3-4, but not ω-conotoxin MVIIA or ω-conotoxin MVIIC, is able to inhibit the uptake of glutamate by synaptosomes, and this inhibition in turn leads to a decrease in the Ca²⁺-independent release of glutamate. No other polypeptide toxin so far described has this effect. PhTx3-4 and ω-conotoxins MVIIC and MVIIA are blockers of voltage-dependent calcium channels, and they significantly inhibited the capsaicin-induced rise of intracellular calcium [Ca²⁺]_i in spinal cord synaptosomes, which likely reflects calcium entry through voltage-gated calcium channels. The inhibition of the calcium-independent glutamate release by PhTx3-4 suggests a potential use of the toxin to block abnormal glutamate release in pathological conditions such as pain.     

Mechanism of trace hyperpolarization of the action potential of snail giant neurons]

Depolarization current decreases and hyperpolarization current increases the amplitude of tracing hyperpolarization of the neuron action potential. Calcium-defficient solution supresses the tracing depolarization, and turns the rhythmical activity of the neuron into the flashlike one. An increase of outer concentration of potassium ions decreases the tracing depolarization. The latter is suppressed completely when the membrane behaves as a potassium electrode. The suppressing effect of the increase of potassium outer concentration on tracing hyperpolarization decreases with a decrease of calcium ions content in the medium. When an active release of sodium ions from the cell is inhibited with DNP and substitution of sodium ions by lithium ions the tracing hyperpolarization of the action potential is suppressed. The tracing hyperpolarization is also suppressed during the shunting of the electrogenic effect of potassium pump with the outcoming current of chlorine ions. It is suggested that the tracing hyperpolarization of the single action potential is due to the calcium-dependent fraction of electrogenic release of sodium ions from the cell.     

Membrane depolarization selectively inhibits receptor-operated calcium channels in human T (Jurkat) lymphoblasts

Jurkat lymphoblasts were stimulated by a monoclonal antibody against the CD3 membrane antigen and the evoked calcium signal was followed by the intracellular fluorescent calcium indicator indo-1. The technique applied allowed us to separately investigate the stimulus-induced intracellular calcium release and the calcium-influx pathways, respectively. In the same cells membrane potential was estimated by the fluorescent dye diS-C3-(5). The resting membrane potential of Jurkat lymphoblasts under normal conditions was between -55 and -60 mV. Membrane depolarization, obtained by increasing external K+ concentration, removing external Cl-, or by increasing the Na+/K+ leak permeability with gramicidin or PCMBS, did not induce calcium influx in the resting cells and did not influence the CD3 receptor-mediated internal calcium release, while strongly inhibited the receptor-mediated calcium influx pathway. Half-maximum inhibition of this calcium influx was observed at membrane potential values of about -35 to -40 mV and this inhibition did not depend on the external calcium concentration varied between 5 and 2500 microM. Membrane hyperpolarization by valinomycin did not affect either component of the calcium signal. The observed selective inhibition of the receptor-operated calcium influx pathway by membrane depolarization is probably an important modulator of calcium-dependent cell stimulation.     

Glutamate receptors modulate sodium-dependent and calcium-independent vitamin C bidirectional transport in cultured avian retinal cells

Vitamin C is transported in the brain by sodium vitamin C co‐transporter 2 (SVCT‐2) for ascorbate and glucose transporters for dehydroascorbate. Here we have studied the expression of SVCT‐2 and the uptake and release of [¹⁴C] ascorbate in chick retinal cells. SVCT‐2 immunoreactivity was detected in rat and chick retina, specially in amacrine cells and in cells in the ganglion cell layer. Accordingly, SVCT‐2 was expressed in cultured retinal neurons, but not in glial cells. [¹⁴C] ascorbate uptake was saturable and inhibited by sulfinpyrazone or sodium‐free medium, but not by treatments that inhibit dehydroascorbate transport. Glutamate‐stimulated vitamin C release was not inhibited by the glutamate transport inhibitor l ‐β‐threo‐benzylaspartate, indicating that vitamin C release was not mediated by glutamate uptake. Also, ascorbate had no effect on [³H] d ‐aspartate release, ruling out a glutamate/ascorbate exchange mechanism. 2‐Carboxy‐3‐carboxymethyl‐4‐isopropenylpyrrolidine (Kainate) or NMDA stimulated the release, effects blocked by their respective antagonists 6,7‐initroquinoxaline‐2,3‐dione (DNQX) or (5R,2S)‐(1)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine hydrogen maleate (MK‐801). However, DNQX, but not MK‐801 or 2‐amino‐5‐phosphonopentanoic acid (APV), blocked the stimulation by glutamate. Interestingly, DNQX prevented the stimulation by NMDA, suggesting that the effect of NMDA was mediated by glutamate release and stimulation of non‐NMDA receptors. The effect of glutamate was neither dependent on external calcium nor inhibited by 1,2‐bis (2‐aminophenoxy) ethane‐N′,N′,N′,N′,‐tetraacetic acid tetrakis (acetoxy‐methyl ester) (BAPTA‐AM), an internal calcium chelator, but was inhibited by sulfinpyrazone or by the absence of sodium. In conclusion, retinal cells take up and release vitamin C, probably through SVCT‐2, and the release can be stimulated by NMDA or non‐NMDA glutamate receptors.     

A Ewing's Sarcoma Cell Line Showing Some, but Not All, of the Traits of a Cholinergic Neuron

Abstract: The Ewing's sarcoma cell line ICB 112 was examined in detail for a cholinergic phenotype. Choline acetyltransferase activity (12.3 ± 2.9 nmol/h/mg of protein) was associated with the presence of multiple mRNA species labeled with a human choline acetyltransferase riboprobe. Choline was taken up by the cells by a high-affinity, hemicholinium-3-sensitive transporter that was partially inhibited when lithium replaced sodium in the incubation medium; the choline taken up was quickly incorporated into both acetylcholine and phosphorylcholine. High-affinity binding sites for vesamicol, an inhibitor of vesicular acetylcholine transport, were also present. The mRNAs for synaptotagmin (p65) and the 15-kDa proteolipid were readily detected and were identical in size to those observed in cholinergic regions of the human brain. Cumulative acetylcholine efflux was increased by raising the extracellular potassium level or the addition of a calcium ionophore, but the time course of stimulated efflux was slow and persistent. These results show that this morphologically undifferentiated cell line is capable of acetylcholine synthesis and expresses markers for synaptic vesicles as well as proteins implicated in calcium-dependent release but lacks an organized release mechanism.     

The induction of kin genes in cold-acclimating Arabidopsis thaliana. Evidence of a role for calcium

The involvement of calcium signaling during cold-induction of the kin genes of Arabidopsis thaliana (L.) Heynh. was examined. Treatments with chemicals which either chelate extracellular calcium (EGTA) or block the plasma-membrane calcium channels (La³⁺, Gd³⁺) inhibited cold acclimation as well as kin gene expression. Ruthenium red, an inhibitor of calcium release from intracellular stores partially inhibited kin gene expression and development of freezing tolerance. An inhibitor of calcium-dependent protein kinases (CDPKs) and calmodulin prevented cold acclimation as well as the cold induction of kin genes. Using restriction fragment length polymorphism-coupled domain-directed differential display, five CDPK clones were identified which showed differential regulation by cold. The amplified fragments showed homology to known plant CDPKs. The involvement of calcium and calcium-binding proteins in cold acclimation of A. thaliana is discussed. Received: 28 November 1996 / Accepted: 5 May 1997