Properties of the caffeine-sensitive intracellular calcium stores in mammalian neurons

Using indo-1- and fura-2-based microfluorometry for measuring the cytoplasmic free calcium concentration ([Ca²⁺] _in ), the properties of caffeine-induced Ca²⁺ release from internal stores were studied in rat cultured central and peripheral neurons, including dorsal root ganglion (DRG) neurons, neurons from then. cuneatus, CA1 and CA3 hippocampal regions, and pyramidal neocortical neurons. Under resting conditions, the Ca²⁺ content of internal stores in DRG neurons was high enough to produce caffeine-triggered [Ca²⁺] _in transients. Prolonged exposure of caffeine depleted the caffeine-sensitive stores of releasable Ca²⁺; the degree of this depletion depended on caffeine concentration. The depletion of the caffeine-sensitive internal stores to some extent was linked to calcium extrusion via La³⁺-sensitive plasmalemmal Ca²⁺-ATPases. Caffeine-induced Ca²⁺ release deprived internal stores in DRG neurons, but they refilled themselves spontaneously within 10 min. Pharmacological manipulation with caffeine-sensitive stores interferred with the depolarization-induced [Ca²⁺] _in transients. In the presence of low caffeine concentration (0.5–1.0 mM) in the extracellular solution, the rate of rise of the depolarization-triggered [Ca²⁺] _in transients significantly increased (by a factor of 2.15 ± 0.29) suggesting the occurrence of Ca²⁺-induced Ca²⁺ release. When the caffeine-sensitive stores were emptied by prolonged application of caffeine, the amplitude and rate of rise of the depolarization-induced [Ca²⁺] _in transients decreased. These findings suggest the involvement of internal caffeine-sensitive calcium stores in generation of calcium signal in sensory neurons. In contrast, in all types of central neurons tested the resting Ca²⁺ content of internal stores was low, but the stores could be charged by transmembrane Ca²⁺ entry through voltage-operated calcium channels. After charging, the stores in central neurons spontaneously lost releasable calcium content and within 10 min they became completely empty again. We suggest that internal Ca²⁺ stores in peripheral and central neurons, although having similar pharmacological characteristics, handle Ca²⁺ ions in a different manner. Calcium stores in sensory neurons are continuously filled by releasable calcium and after discharging they can be spontaneously refilled, whereas in central neurons internal calcium stores can be charged by releasable calcium only transiently. Caffeine-evoked [Ca²⁺] _in transients in all types of neurons were effectively blocked by 10 mM ryanodine, 5 mM procaine, 10 mM dantrolene, or 0.5 mM Ba²⁺, thus sharing the basic properties of the Ca²⁺-induced Ca²⁺ release from endoplasmic reticulum.Neirofiziologiya/Neurophysiology, Vol. 26, No. 1, pp. 16–25, January–February, 1994.     

Activation and deactivation of sarcoplasmic reticulum calcium release channels: Molecular dissection of mechanisms via novel semi-synthetic ryanoids

The plant alkaloids ryanodine and dehydroryanodine are high affinity, biphasic modulators of the intracellularly located, calcium-regulated calcium release channels of a variety of cell types. To date, little is certain about the molecular basis of the interactions that prompt low concentrations of ryanodine (nanomolar to low micromolar) to activate (open) the channels and higher concentrations to deactivate (functionally close) the sarcoplasmic reticulum calcium release channel. In the present study, we approached this question using novel, semi-synthetic C₁₀–O_eq ester derivatives of ryanodine and dehydroryanodine as molecular probes of the ryanodine binding sites on the calcium release channel.Binding affinities of these C₁₀–O_eq ester derivatives of ryanodine and dehydroryanodine with acidic, basic and neutral side chains (K_d values> 53.9 nM, K_d values 0.3–0.7 nM and K_d values 1.3–20.4 nM, compared with 2.3 and 2.8 nM for ryanodine and dehydroryanodine, respectively) were evaluated for their ability to modulate, the patency of the sarcoplasmic reticulum calcium release channel. With the exception of only two derivatives tested to date, all the semi-synthetic C₁₀–O_eq esters selectivelyactivate the Ca²⁺ release channel. That is, they produce no functional closure of the sarcoplasmic reticulum calcium release channels at the highest concentration, that could be tested. Half-maximal concentrations for activation (EC_50act , values) ranged from 0.87–4.2, M, compared with an EC_50act of 1.3 M for ryanodine.     

Calcium Homeostasis and Cone Signaling Are Regulated by Interactions between Calcium Stores and Plasma Membrane Ion Channels

Calcium is a messenger ion that controls all aspects of cone photoreceptor function, including synaptic release. The dynamic range of the cone output extends beyond the activation threshold for voltage-operated calcium entry, suggesting another calcium influx mechanism operates in cones hyperpolarized by light. We have used optical imaging and whole-cell voltage clamp to measure the contribution of store-operated Ca²⁺ entry (SOCE) to Ca²⁺ homeostasis and its role in regulation of neurotransmission at cone synapses. Mn²⁺ quenching of Fura-2 revealed sustained divalent cation entry in hyperpolarized cones. Ca²⁺ influx into cone inner segments was potentiated by hyperpolarization, facilitated by depletion of intracellular Ca²⁺ stores, unaffected by pharmacological manipulation of voltage-operated or cyclic nucleotide-gated Ca²⁺ channels and suppressed by lanthanides, 2-APB, MRS 1845 and SKF 96365. However, cation influx through store-operated channels crossed the threshold for activation of voltage-operated Ca²⁺ entry in a subset of cones, indicating that the operating range of inner segment signals is set by interactions between store- and voltage-operated Ca²⁺ channels. Exposure to MRS 1845 resulted in ∼40% reduction of light-evoked postsynaptic currents in photopic horizontal cells without affecting the light responses or voltage-operated Ca²⁺ currents in simultaneously recorded cones. The spatial pattern of store-operated calcium entry in cones matched immunolocalization of the store-operated sensor STIM1. These findings show that store-operated channels regulate spatial and temporal properties of Ca²⁺ homeostasis in vertebrate cones and demonstrate their role in generation of sustained excitatory signals across the first retinal synapse.     

Mechanisms of cytoplasmic calcium signalling in cerebellar bergmann glial cells

Mechanisms underlying intracellular calcium signals in Bergmann glial cells evoked by various neurotransmitters were investigated in experiments on cerebellar slices acutely isolated from 30-day-old mice. [Ca²⁺] _in values were measured by means of a Ca²⁺-sensitive fluorescent probe fura-2. Extracellular application of ATP (10–100 µM), histamine (10–100 µM), or noradrenaline (or adrenaline, 0.1–10.0 µM) caused a temporary increase in cytoplasmic Ca²⁺ concentrations. The effect persisted in Ca²⁺-free extracellular solution and was blocked with thapsigargin (500 nM) or a specific blocker of the inositol-1,4,5-trisphosphate-sensitive intracellular channels heparin. Based on the pharmacological analysis, we postulate the involvement of P₂ purinoreceptors, ₁-adrenoreceptors, and H₁ histamine receptors in an agonist-activated increase in [Ca²⁺] _in in Bergmann glia. Thus, ATP, monoamines, or histamine induce calcium signal generation in Bergmann glial cells via activation of Ca²⁺ release from the inositol-1,4,5-trisphosphate-sensitive internal stores.Neirofiziologiya/Neurophysiology, Vol. 26, No. 6, pp. 417–419, November–December, 1994.     

Physical Link and Functional Coupling of Presynaptic Calcium Channels and the Synaptic Vesicle Docking/Fusion Machinery

N- and P/Q-type calcium channels are localized in high density in presynaptic nerve terminals and are crucial elements in neuronal excitation–secretion coupling. In addition to mediating Ca²⁺ entry to initiate transmitter release, they are thought to interact directly with proteins of the synaptic vesicle docking/fusion machinery. As outlined in the preceding article, these calcium channels can be purified from brain as a complex with SNARE proteins which are involved in exocytosis. In addition, N-type and P/Q-type calcium channels are co-localized with syntaxin in high-density clusters in nerve terminals. Here we review the role of the synaptic protein interaction (synprint) sites in the intracellular loop II–III (L_II–III) of both _1B and _1A subunits of N-type and P/Q-type calcium channels, which bind to syntaxin, SNAP-25, and synaptotagmin. Calcium has a biphasic effect on the interactions of N-type calcium channels with SNARE complexes, stimulating optimal binding in the range of 10–20 M. PKC or CaM KII phosphorylation of the N-type synprint peptide inhibits interactions with native brain SNARE complexes containing syntaxin and SNAP-25. Introduction of the synprint peptides into presynaptic superior cervical ganglion neurons reversibly inhibits EPSPs from synchronous transmitter release by 42%. At physiological Ca²⁺ concentrations, synprint peptides cause an approximate 25% reduction in transmitter release of injected frog neuromuscular junction in cultures, consistent with detachment of 70% of the docked vesicles from calcium channels based on a theoretical model. Together, these studies suggest that presynaptic calcium channels not only provide the calcium signal required by the exocytotic machinery, but also contain structural elements that are integral to vesicle docking, priming, and fusion processes.     

Atrial natriuretic factor (ANF) effects on L-, N-, and P/Q-type voltage-operated calcium channels

1. We have previously reported that atrial natriuretic factor (ANF) decreases neuronal norepinephrine (NE) release. The mechanism that mediates NE release from presynaptic membrane to synaptic cleft is a strongly calcium-dependent process. The modulator effect of ANF may be related to modifications in calcium influx at the presynaptic nerve ending by interaction with voltage-operated calcium channels (VOCCs).2. On this basis we investigated the effects of ANF on K⁺-induced ⁴⁵Ca²⁺ uptake and evoked neuronal NE release in the presence of specific L-, N-, and P/Q-type calcium channel blockers in the rat hypothalamus.3. Results showed that ANF inhibited K⁺-induced ⁴⁵Ca²⁺ uptake in a concentration-dependent fashion. Concentration–response curves to VOCC blockers nifedipine (NFD, L-type channel blocker), -conotoxin GVIA (CTX, N-type channel blocker), and -agatoxin IVA (AGA, P/Q-type channel blocker) showed that all the blockers decreased NE release. Incubation of ANF plus NFD showed an additive effect as compared to NFD or ANF alone. However, when the hypothalamic tissue was incubated in the presence of ANF plus CTX or AGA there were no differences in neuronal NE release as compared to calcium channel blockers or ANF alone.4. These results suggest that ANF decreases NE release by an L-type calcium channel independent mechanism by inhibiting N- and/or P/Q-type calcium channels at the neuronal presynaptic level. Thus, ANF modulates neuronal NE release through different mechanisms involving presynaptic calcium channel inhibition.     

Basic mechanisms determining the characteristics of calcium signals in nerve cells

The paper summarizes recent data about the mechanisms that determine the kinetics and amplitude of transient elevations in the intracellular level of free calcium (calcium signals) in excitable cells. The relative role of various types of voltage-operated calcium channels, fast cytosolic buffering, active accumulation in intracellular stores, and extrusion of ions from the cell are discussed. New technical approaches enabling resolution of these questions are described.Neirofiziologiya/Neurophysiology, Vol. 26, No. 1, pp. 5–8, January–February, 1994.     

Calcium influx and intracellular calcium release in anti-CD3 antibody-stimulated and thapsigargin-treated human T lymphoblasts

Summary Jurkat and MOLT-4 cultured T lymphoblasts were loaded with low concentrations (30–50 m) of indo-1 and with high concentrations (3.5–4.5mm) of quin-2, respectively, in order to follow the activation of calcium transport pathways after stimulation of the cells by a monoclonal antibody against the T cell antigen receptor (aCD3), or after the addition of thapsigargin, a presumed inhibitor of endoplasmic reticulum calcium pump. In the indo-1 loaded cells the dynamics of the intracellular calcium release and the calcium influx could be studied, while in the quin-2 overloaded cells the changes in cytoplasmic free calcium concentration ([Ca²⁺] _i ) were strongly buffered and the rate of calcium influx could be quantitatively determined. We found that in Jurkat lymphoblasts, in the absence of external calcium, both aCD3 and thapsigargin induced a rapid calcium release from internal stores, while upon the readdition of external calcium an increased rate of calcium influx could be observed in both cases, aCD3 and thapsigargin released calcium from the same intracellular pools. The calcium influx induced by either agent was of similar magnitude and had a nonadditive character if the two agents were applied simultaneously. As demonstrated in quin-2 overloaded cells, a significant initial rise in [Ca²⁺] _i or a pronounced depletion of internal calcium pools was not required to obtain a rapid calcium influx. The activation of protein kinase C by phorbol ester abolished the internal calcium release and the calcium influx induced by aCD3, while having only a small effect on these phenomena when evoked by thapsigargin. Membrane depolarization by gramicidin inhibited the rapid calcium influx in both aCD3- and thapsigargin-treated cells, although it did not affect the internal calcium release produced by either agent. In MOLT-4 cells, which have no functioning antigen receptors, aCD3 was ineffective in inducing a calcium signal, while thapsigargin produced similar internal calcium release and external calcium influx to those observed in Jurkat cells.     

Excitatory Amino Acid-Mediated Cytotoxicity and Calcium Homeostasis in Cultured Neurons

Abstract: A large body of evidence suggests that disturbances of Ca²⁺ homeostasis may be a causative factor in the neurotoxicity induced by excitatory amino acids (EAAs). The route or routes by which an increase in intracellular calcium concentration ([Ca²⁺]_i) is mediated in vivo are presently not clarified. This may partly reflect the complexity of intact nervous tissue in combination with the relative unspecific action of the available “calcium antagonists,” e.g., blockers of voltage-sensitive calcium channels. By using primary cultures of cortical neurons as a model system, it has been found that all EAAs stimulate increases in [Ca²⁺]_i but via different mechanisms. By using the drug dantrolene, it has been shown that 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionate (AMPA) apparently exclusively stimulates Ca²⁺ influx through agonist-operated calcium channels and voltage-operated calcium channels. Increased [Ca²⁺]_i due to exposure to kainate (KA) is for the major part caused by influx, as in the case of AMPA, but a small part of the increase in [Ca²⁺]_i may be attributed to a release of Ca²⁺ from intracellular stores. Quisqualate (QA) stimulates Ca²⁺ release from an intracellular store that is independent of Ca²⁺ influx; presumably this store is activated by inositol phosphates. The increase in [Ca²⁺]_i due to exposure to glutamate or N-methyl-d -aspartate (NMDA) may be compartmentalized into three components, one of which is related to influx and the other two to Ca²⁺ release from internal stores. Only one of the latter stores is dependent on Ca²⁺ influx with regard to release of Ca²⁺, whereas the other is activated by some other second messengers or, alternatively, directly coupled to the receptor. In muscles dantrolene is known to inhibit Ca²⁺ release from the sarcoplasmic reticulum, and also in neurons dantrolene inhibits an equivalent release from one or more hitherto unidentified internal Ca²⁺ pool(s). By using this drug it has been possible to show to what extent these Ca²⁺ stores are involved in the toxicity observed subsequent to exposure to the EAAs. It turned out that dantrolene, even under conditions allowing Ca²⁺ influx, inhibited toxicity induced by QA, NMDA, and glutamate, whereas that induced by AMPA or KA was unaffected. In combination with the findings that dantrolene inhibited release from the intracellular stores activated by QA, NMDA, and glutamate, it may be concluded that Ca²⁺ influx per se is not the primary event causing toxicity following exposure to these EAAs in these neurons. However, it may certainly be involved in the cases of toxicity induced by AMPA and KA. Finally, it should be pointed out that this model only serves as a much simplified working hypothesis and that the situation in vivo is much more complex.     

Modeling the Calcium Signaling in Stomatal Guard Cells under the Action of Abscisic Acid

A theoretical model of calcium signaling is presented that simulates oscillations of cytoplasmic calcium concentration ([Ca²⁺]_cyt) in stomatal guard cells under the action of abscisic acid. The model is based on the kinetics of inositol 1,4,5-trisphosphate-sensitive calcium channels of endoplasmic reticulum and cyclic ADP-ribose-sensitive calcium channels of the tonoplast. The operation of two energy-dependent pumps—the Ca²⁺-ATPase of the endoplasmic reticulum and the Ca²⁺/H⁺ antiporter of the tonoplast—is also included in the model. It is shown that the removal of excessive Ca²⁺ from the cytoplasm by the tonoplast Ca²⁺/H⁺ antiporter is the main factor accounting for generation of [Ca²⁺]_cyt oscillations at a wide range of ABA concentrations (0.01–1 M). The long period of [Ca²⁺]_cyt oscillations in plant cells is explained by a slow release from inhibition of inositol 1,4,5-trisphosphate-gated calcium channels.     

Changes in the ionic mechanisms of electrical excitability in the somatic membrane of rat dorsal root ganglion neurons during ontogenesis. Correlations between inward current densities

Correlations between densities of various types of inward currents in the somatic membrane of dorsal root ganglion neurons were studied in three different rat age groups: 5–9 days, 45 days, and 90 days. A linear relationship was found in neurons with "slow" tetrodotoxin-sensitive sodium current between the densities of high-threshold calcium current and "slow" sodium current (Bravias-Pearson's correlation coefficient: r=0.84 and 0.70 for n₁=16 and n₂=28, respectively). No such correlation was observed in neurons with low-threshold calcium inward current. Cells with only two types of channel — "fast" sodium and high-threshold calcium — present in their somatic membrane manifested an inverse correlation (r=–0.48, where n₄=95) between the densities of transmembrane currents passing through these channels. No inverse relationship was observed in the density of "fast" sodium and high-threshold calcium currents in neurons with tetradotoxinresistant "slow" sodium and/or low threshold calcium channels.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 18, No. 6, pp. 820–827, November–December, 1986.     

Calcium signals activated by ghrelin and D-Lys-GHRP-6 ghrelin antagonist in developing dorsal root ganglion glial cells

Ghrelin is a hormone regulating energy homeostasis via interaction with its receptor, GHSR-1a. Ghrelin activities in dorsal root ganglia (DRG) cells are unknown. Herein we show that ghrelin induces a change of cytosolic calcium concentration in both glia and neurons of embryonic chick DRG. Both RT-PCR and binding studies performed with fluorescent ghrelin in the presence of either unlabeled ghrelin or GHSR-1a antagonist D-Lys³-GHRP-6, indicate that DRG cells express GHSR-1a. In glial cells the response is characterized by a rapid transient rise in [Ca²⁺]_i followed by a long lasting rise. The calcium elevation is dependent on calcium release from thapsigargin-sensitive intracellular stores and on activation of two distinct Ca²⁺ entry pathways, a receptor activated calcium entry and a store operated calcium entry. Surprisingly, D-Lys³-GHRP-6 exerts several activities in the absence of exogenous ghrelin: (i) it activates calcium release from thapsigargin-sensitive intracellular stores and calcium entry via voltage-operated channels in non-neuronal cells; (ii) it inhibits calcium oscillations in non-neuronal cells exhibiting spontaneous Ca²⁺ activity and iii) it promotes apoptosis of DRG cells, both neurons and glia. In summary, we provide the first evidence for ghrelin activity in DRG, and we also demonstrate that the widely used D-Lys³-GHRP-6 ghrelin antagonist features ghrelin independent activities.     

Classes and mechanisms of calcium waves

The best known calcium waves move at about 5–30 μm/s (at 20°C) and will be called fast waves to distinguish them from slow (contractile) ones which move at 0.1-1 μm/s as well as electrically propagated, ultrafast ones. Fast waves move deep within cells and seem to underlie most calcium signals. Their velocity and hence mechanism has been remarkably conserved among all or almost all eukaryotic cells. In fully active (but not overstimulated) cells of all sorts, their mean speeds lie between about 15–30 μm/s at 20°C. Their amplitudes usually lie between 3–30 μM and their frequencies from one per 10–300 s. They are propagated by a reaction diffusion mechanism governed by the Luther equation in which Ca²⁺ ions are the only diffusing propagators, and calcium induced calcium release, or CICR, the only reaction; although this reaction traverses various channels which are generally modulated by IP₃ or cADPR. However, they may be generally initiated by a second, lumenal mode of CICR which occurs within the ER. Moreover, they are propagated between cells by a variety of mechanisms. Slow intracellular waves, on the other hand, may be mechanically propagated via stretch sensitive calcium channels.     

Single chloride channels in colon mucosa and isolated colonic enterocytes of the rat

Summary Chloride channels from rat colonic enterocytes were studied using the patch-clamp technique. After removal of mucus, inside-out patches were excised from the apical membrane of intact epithelium located at the luminal surface. They contained spontaneously switching Cl^– channels with a conductance of 35–40 pS. The channels were blocked reversibly by anthracene-9-carboxylic acid (1mm).In excised patches from single enterocytes, isolated by calcium removal, the Cl^– channels were studied in more detail. TheI–V relation was linear between ±80 mV. The selectivity was I^–>Br^–>Cl^–=NO ₃ ^– >F^–=HCO ₃ ^– .Thirty pS Cl^– channels were also found on the basolateral membrane of crypts isolated by brief calcium removal. TheI–V curve of these Cl^– channels was also linear.The results provide direct evidence for the existence of Cl^– channels in the apical membrane of surface cells in colonic mucosa. The properties of these channels are similar to those previously observed when incorporating membrane vesicles into planar lipid bilayers. Both results support the validity of the theoretical models describing intestinal secretion.     

Voltage-gated ion channels of inflowing current expressed in Xenopus oocytes after injection of rat brain mRNA

The expression of two types of voltage-gated ion channels of the inflowing current ("fast" sodium channels, sensitive to tetrodotoxin, and high-threshold calcium channels) was detected by electrophysiological methods in the membrane ofXenopus oocytes, after injection of poly(A)⁺-mRNA from the brains of 18- to 20-day-old rats. When Cd²⁺ (200 µmoles/liter) was added to the extracellular solution, the barium current through the expressed calcium channels was completely suppressed, but no sensitivity to D-600 (20 µmoles/liter) and nitrendipine (50 µmoles/liter) was exhibited. A peptide blocker of the high-threshold calcium channels of the neuron membrane, -conotoxin GVIA, in a concentration of 1 µmole/liter led to 20–40 min suppression of the barium current expressed in the oocyte. Steady-state inactivation of this current could be described by the Boltzman formula, using the values of the half-inactivation potential V_1/2=–50 mV and the steepness factor k=14 mV. It is concluded that in potential-dependent and pharmacological properties, the calcium channels expressed in the oocyte, despite the absence of any appreciable time-dependent inactivation, most resemble the high-threshold inactivatable (HTI- or N-type) calcium channels of the neuron membrane.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 3, pp. 344–353, May–June, 1991.     

Cardiotoxicity of calmidazolium chloride is attributed to calcium aggravation, oxidative and nitrosative stress, and apoptosis

The intracellular calcium concentration ([Ca]_i) regulates cell viability and contractility in myocardial cells. Elevation of the [Ca]_i level occurs by entry of calcium ions (Ca²⁺) through voltage-dependent Ca²⁺ channels in the plasma membrane and release of Ca²⁺ from the sarcoplasmic reticulum. Calmidazolium chloride (CMZ), a subgroup II calmodulin antagonist, blocks L-type calcium channels as well as voltage-dependent Na⁺ and K⁺ channel currents. This study elaborates on the events that contribute to the cytotoxic effects of CMZ on the heart. We hypothesized that apoptotic cell death occurs in the cardiac cells through calcium accumulation, production of reactive oxygen species, and the cytochrome c-mediated PARP activation pathway. CMZ significantly increased the production of superoxide (O₂^•–) and nitric oxide (NO) as detected by FACS and confocal microscopy. CMZ induced mitochondrial damage by increasing the levels of intracellular calcium, lowering the mitochondrial membrane potential, and thereby inducing cytochrome c release. Apoptotic cell death was observed in H9c2 cells exposed to 25 μM CMZ for 24 h. This is the first report that elaborates on the mechanism of CMZ-induced cardiotoxicity. CMZ causes apoptosis by decreasing mitochondrial activity and contractility indices and increasing oxidative and nitrosative stress, ultimately leading to cell death via an intrinsic apoptotic pathway.     

Phosphorus release from resuspended sediment in the shallow and wind-exposed Lake Arresø, Denmark

Wind-induced sediment resuspension occurs frequently in the shallow and eutrophic Lake Arresø, Denmark. The impact of resuspension on internal phosphorus loading was investigated by laboratory experiments studying P-release from the undisturbed sediment surface and by experiments simulating resuspension events.Phosphorus release from undisturbed sediment sampled in May and August was 12 mg and 4 mg m^–2 d^–1, respectively. During experimental simulation of resuspension, soluble reactive phosphate (SRP) increased by 20–80 µg l^–1, which indicates that a typical resuspension event in the lake would be accompanied by the release of 150 mg SRP m^–2. The internal P loading induced by resuspension is estimated to be 60–70 mg m^–2 d^–1, or 20–30 times greater than the release from undisturbed sediment.SRP release during simulation of resuspension was mainly dependent on the equilibrium conditions in the water column and was basically independent of the increase in suspended solids and the duration of resuspension. A second simulation of resuspension conducted 26 hours later, did not result in any further release of SRP from sediment sampled in May. In contrast, there was an additional SRP release from sediment sampled in August, indicating that an exchangable P pool, capable of altering equilibrium conditions, is built up between resuspension events.It is concluded that resuspension, by increasing the P flux between sediment and water, plays a major role in the maintenance of the high nutrient level in Lake Arresø. A relatively high release rate is maintained during resuspension because of the low Fe:P ratio and the high concentration of NH₄Cl-extractable P in the sediment.     

Differentiated expression inXenopus oocytes of calcium channels from rat cerebellar and forebrain neurons

Calcium channels were expressed inXenopus laevis oocytes by means of matrix RNA (mRNA) extracted from the cerebellum (RNAc) and forebrain (RNAfb). In these oocytes, inward barium currents,I _Ba, evoked by 40 mM Ba²⁺ were investigated using a double microelectrode technique. Currents expressed after injection of both RNAc and RNAfb (further referred to as RNAc- and RNAfb-expressed currents) showed a voltage-dependent characteristic typical of high-threshold calcium channels of mammalian neurons. The threshold of activation was about –40 mV, the maximum amplitude was observed at +20 mV and reversal potential at +60 mV. In both groups of oocytes, no expression of low- or high-threshold calcium channels of other types was observed. Although in both cases the expression ofI _Ba had similar macrokinetics, characteristics of their stationary inactivation differed. The half-inactivation potential ranged between –32 and –16 mV, and the slope factor was 28 and 16.6 mV in RNAfb- and RNAc-injected oocytes, respectively. In both cases,I _Ba were insensitive to dihydropyridines; however their relation to other pharmacological agents was different. RNAfb-expressedI _Ba was completely blocked by Cd²⁺ (K _d=10 µM) and depressed up to 70% by -conotoxin (1 µM), being insensitive to either whole spider toxin fromAgelenopsis aperta venom or to its FTX fraction. On the contrary, RNAc-expressedI _Ba was more sensitive to Cd²⁺ (K _d=0.1 µM), stable to -conotoxin, and suppressed up to 75–90% by wholeA. aperta toxin in a dilution of 1:10000, and by FTX at a concentration of 0.5 µM. The findings allow us to suggest that the forebrain and cerebellum of mammals are the structures, whose mRNA differ and provide predominant expression of voltage-dependent calcium channels of N- and P-types, respectively.Neirofiziologiya/Neurophysiology, Vol. 26, No. 6, pp. 427–436, November–December, 1994.     

Interaction between perchlorate and nifedipine on insulin secretion from mouse pancreastic islets

In order to elucidate the mechanisms responsible for the stimulatory effect of perchlorate (ClO ₄ ^– ) on insulin secretion, we have investigated the interaction between this chaotropic anion and the organic calcium antagonist nifedipine. This drug, known as a blocker of L-type calcium channels, was chosen as a tool to test the idea that ClO ₄ ^– acts on insulin secretion by stimulating the gating of voltage-controlled Ca²⁺ channels. ClO ₄ ^– amplified the stimulatory effect of D-glucose on insulin release from perfused pancreas (first and second phases) as well as from isolated islets incubated in static incubations for 60 min. This indicates that ClO ₄ ^– amplifies physiologically regulated insulin secretion. Nifedipine reduced D-glucose-induced (20 mM) insulin release in a dose-dependent manner with half-maximum effect at about 0.8 M and apparent maximum effect at 5 M nifedipine. In the presence of 20 mM D-glucose, the inhibitory effects of 0.5, 1 or 5 M nifedipine were only slightly, if at all, counteracted by perchlorate. When 12 mM ClO ₄ ^– and 20 mM D-glucose were combined, calculation of the specific effect of ClO ₄ ^– revealed that nifedipine produced almost maximum inhibition already at 0.05 M. Thus, the perchlorate-induced amplification of D-glucose-stimulated insulin release shows higher sensitivity to nifedipine than the D-glucose-effect as such. This supports the hypothesis that perchlorate primarily affects the voltage-sensitive L-type calcium channel in the -cell.     

Effect of substances blocking calcium channels (verapamil,D-600, manganese ions) on transmitter release from motor nerve endings in frog muscle

Experiments on isolated frog nerve-muscle preparations showed that manganese ions (0.4–5.0 mM) inhibit evoked transmitter release by reducing the quantum composition of the end-plate potentials, and they intensify spontaneous transmitter release to a certain extent by increasing the frequency of miniature potentials. Verapamil (1 · 10^–6–5·10^–5 g/ml) and D-600 (2.5·10^–5 g/ml), by contrast with manganese ions, do not inhibit evoked release, but also intensify spontaneous release of the transmitter. All the agents tested prevent the potentiating effect of imidazole (3 mM). During repetitive stimulation, verapamil disturbs action potential generation in the motor nerve. Manganese ions had no such action. It is concluded that between the calcium channels of motor nerve endings and the calcium channels of heart muscle or the neuron soma there are molecular differences, expressed as sensitivity to the blocking action of verapamil and D-600.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 9, No. 4, pp. 415–422, July–August, 1977.