Premotor nonspiking neurons regulate coupling among motoneurons that innervate overlapping muscle fiber population

Recent work indicated that co-activity of different motoneurons (MNs) in the leech can be regulated through a network that is centered on a pair of nonspiking (NS) neurons. Here, we investigate whether this effect generalizes to different types of MNs that display differential co-activity patterns in different motor behaviors: the dorsal longitudinal excitors DE-3 and the dorsal and ventral excitors MN-L. The data indicates that both motoneurons are coupled to the NS neurons through rectifying junctions that are activated when the motoneuron membrane potential becomes more negative than that of the NS, and that they exert an inhibitory synaptic potential on NS via a polysynaptic pathway. In addition, DE-3 and MN-L are linked by junctions that allow mutual excitation but the transmission of excitatory signals from MN-L to DE-3 depended on NS membrane potential. The results support the view that NS neurons can play a central role in orchestrating the co-activity of MNs during motor behaviors.     

Mg2+- ATPase in the fish brain and its ultrastructural localization

Effects of γ-aminobutyric acid (GABA) and ethanol on Mg²⁺-ATPase from mitochondrial and microsomal fractions of the fish brain were studied. GABA (10^-8–10^-4 M) activates microsomal Mg²⁺-ATPase, but has no effect on the mitochondrial enzyme activity. This effect of GABA on the microsomal Mg²⁺-ATPase was absent in the presence of 8% ethanol. Ethanol at 1– 10% concentrations inhibits the basal microsomal Mg²⁺-ATPase and has no effect on the mitochondria enzyme. Using cytochemical technique, Mg²⁺-ATPase was revealed both in neurons and in glial cells. The ethanol-sensitive Mg²⁺-ATPase is located in the area of synaptic junctions and is bound to plasma, vesicular, and smooth endoplasmic reticulum membranes.     

Is the Mg2+-ATP-dependent proton pumping activity of the synaptic vesicles a factor involved in the cerebral hypoxia?

The changes in the Mg²⁺-dependent V-type ATPase activity and the Mg²⁺-ATP-dependent H⁺ pumping activity of the synaptic vesicles from the cerebral cortex of rats submitted to intermittent chronic (4 weeks) mild or severe hypoxia were evaluated. The adaptation to the chronic severe hypoxia increases both the ATPase and the H⁺ pumping activities which are inhibited by NEM with an exponential relationship between the IC₅₀ values and the in vivo O₂ concentration. The Mg²⁺-dependent increase in H⁺ pumping activity of synaptic vesicles from the rats subjected to in vivo chronic hypoxia may be antagonized by nigericin (dissipating ΔpH) and by FCCP (dissipating ΔpH and Δ_ΨSV). In contrast, valinomycin (dissipating the Δ_ΨSV and facilitating an enhancement in ΔpH) increases in vitro the H⁺ pumping activity that is inhibited by the addition of high concentration of K gluconate (reducing the rate of K⁺ efflux). The preincubation of vesicles from hypoxic rats with FCCP, but not with nigericin, inhibits the valinomycin-increased H⁺ pumping activity.l-glutamate increases the H⁺ pumping activity in synaptic vesicles from the cerebral cortex of chronic hypoxic rats, whereas other amino acids (i.e.,l-aspartate andl-homocysteate) and glutamate analogs (i.e., quisqualate and ibotenate) are ineffective. The adaptation to both chronic intermittent severe hypoxia and in vivo treatment with posatireline causes a decrease in the Mg²⁺-ATPase activity consistent with the decrease in the H⁺ pumping one of the synaptic vesicles. The addition of nigericin into incubation medium magnifies the decrease in the H⁺ pumping activity, while the addition of FCCP is ineffective, suggesting that the treatment with posatireline interferes with the Δ_ΨSV component in the of the synaptic vesicles from rats submitted to chronic hypoxia. The results of the in vivo and in vitro experiments suggest that in the synaptic vesicles from hypoxic rats the Δ_ΨSV component in may be most effective in increasing the Mg²⁺-ATP-dependent H⁺ pumping activity.     

Synaptic function and modulation of glycine receptor channels in the hypoglossal nucleus

In this review, we discuss the function and modulation of chloride-selective glycine receptor (GlyR) channels, some genetic diseases originated from dysfunction of GlyRs, and modulation of glycinergic synapses by intracellular calcium (Ca²⁺) with particular attention on the motoneurons of the hypoglossal nucleus. This motor nucleus is a brainstem structure implicated in the command of coordinated movements during oral behavioral phenomena, including feeding, drinking, grooming, and respiration. In this nucleus, more than 90% of its cells are motoneurons. These hypoglossal motoneurons (HMs) are involved in a variety of motor functions and exhibit two remarkable features: (i) a low endogenous Ca²⁺ buffering capacity, which determines the rapid dynamics of cytosolic intracellular Ca²⁺, and (ii) powerful glycinergic inputs, which determine the main inhibitory drive on the above cells in adult animals. Glycine receptors belong to the superfamily of Cys-loop ligand-gated ion channels. They are capable of forming functional homo-or heteromeric chloride-selective channels. Dysfunction of GlyRs results in a genetic neurological motor disorders, including hyperekplexia. These diseases originate from mutations in the GlyR gene, leading to a decrease in single channel conductance, a lower affinity to the neurotransmitter, or a low level of GlyR expression. The function of glycinergic synapses is modulated during developmental changes and strictly controlled by several feedback mechanisms at pre-and post-synaptic levels. The developmental modulation consists in switch in the GlyR subunit composition and change in the chloride homeostasis during the synaptic maturation and formation of inhibitory networks. Retrograde signalling plays an important role in the synaptic function of HMs; it provides post-synaptic neurons with efficient tools for controlling pre-synaptic afferents. Glycine receptors and glycinergic synapses are also regulated by intracellular Ca²⁺. The mechanisms of these modulations are discussed. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 338–349, July–October, 2007.     

Intracellular Mg<Superscript><Emphasis Type="Bold">2+</Emphasis></Superscript> Influences Both Open and Closed Times of a Native Ca<Superscript><Emphasis Type="Bold">2+</Emphasis></Superscript>-activated BK Channel in Cultured Human Renal Proximal Tubule Cells

Effects of intracellular Mg²⁺ on a native Ca²⁺-and voltage-sensitive large-conductance K⁺ channel in cultured human renal proximal tubule cells were examined with the patch-clamp technique in the inside-out mode. At an intracellular concentration of Ca²⁺ ([Ca²⁺]_i) of 10⁻⁵–10⁻⁴ M, addition of 1–10 mM Mg²⁺ increased the open probability (P_o) of the channel, which shifted the P_o –membrane potential (V_m) relationship to the negative voltage direction without causing an appreciable change in the gating charge (Boltzmann constant). However, the Mg²⁺-induced increase in P_o was suppressed at a relatively low [Ca²⁺]_i (10^−5.5–10⁻⁶ M). Dwell-time histograms have revealed that addition of Mg²⁺ mainly increased P_o by extending open times at 10⁻⁵ M Ca²⁺ and extending both open and closed times simultaneously at 10^−5.5 M Ca²⁺. Since our data showed that raising the [Ca²⁺]_i from 10⁻⁵ to 10⁻⁴ M increased P_o mainly by shortening the closed time, extension of the closed time at 10^−5.5 M Ca²⁺ would result from the Mg²⁺-inhibited Ca²⁺-dependent activation. At a constant V_m, adding Mg²⁺ enhanced the sigmoidicity of the P_o–[Ca²⁺]_i relationship with an increase in the Hill coefficient. These results suggest that the major action of Mg²⁺ on this channel is to elevate P_o by lengthening the open time, while extension of the closed time at a relatively low [Ca²⁺]_i results from a lowering of the sensitivity to Ca²⁺ of the channel by Mg²⁺, which causes the increase in the Hill coefficient. M. Kubokawa and Y. Sohma contributed equally to this work.     

News & Notes: Sorption and Desorption of Cobalt by Oscillatoria anguistissima

Oscillatoria anguistissima rapidly adsorbs appreciable amounts of cobalt from the aqueous solutions within 15 min of initial contact with the metal solution. O. anguistissima showed a high sequestration of cobalt at low equilibrium concentrations, and it followed the Freundlich model of adsorption. The adsorption is a strongly pH-dependent and temperature-independent phenomenon. The presence of Mg²⁺ and Ca²⁺ (100–200 ppm) resulted in decline in Co²⁺ adsorption capacity of Oscillatoria biomass. Sulphate and nitrate (0.75–10 mM) drastically reduced the extent of Co²⁺ biosorption. The biosorption of cobalt is an ion-exchange process as the Co²⁺ binding was accompanied by release of a large amounts of Mg²⁺ ions. Na₂CO₃ (1.0 mM) resulted in about 76% desorption of Co²⁺ from the loaded biomass. Received: 30 January 1999 / Accepted: 3 March 1999     

The effect of glucagon on the kinetics of hepatic mitochondrial calcium uptake

Summary Previous work by this and other laboratories has shown that glucagon administration stimulates calcium uptake by subsequently isolated hepatic mitochondria. This stimulation of hepatic mitochondrial Ca²⁺ uptake byin vivo administration of glucagon was further characterized in the present report. Maximal stimulation of mitochondrial Ca²⁺ accumulation was achieved between 6–10 min after the intravenous injection of glucagon into intact rats. Under control conditions, Ca²⁺ uptake was inhibited by the presence of Mg²⁺ in the incubation medium. Glucagon treatment, however, appeared to obliterate the observed inhibition by Mg²⁺ of mitochondrial Ca²⁺ uptake. Kinetic experiments revealed the usual sigmoidicity associated with initial velocity curves for mitochondrial calcium uptake. Glucagon treatment did not alter this sigmoidal relationship. Glucagon treatment significantly increased the V_max for Ca²⁺ uptake from 292±22 to 377±34 nmoles Ca²⁺ /min per mg protein (n=8) but did not affect the K_0.5, (6.5–8.6 μM). Since the major kinetic change in mitochondrial Ca²⁺ uptake evoked by glucagon is an increase in V_max, the enhancement mechanism is likely to be an increase either in the number of active transport sites available to Ca²⁺ or in the rate of Ca²⁺ carrier movement across the mitochondrial membranes.     

Synaptic transmission in sympathetic vasoconstrictor pathways and its modification after injuries

The discharge of vasoconstrictor pathways arising in the CNS is largely unmodified as it passes through the sympathetic ganglia to the vasculature. The underlying synaptic events have been revealed by intracellular recordings from sympathetic paravertebral ganglion cells in the course of ongoing and reflex activity in anesthetized animals, first made in Skok’s Laboratory in Kyiv (Ukraine). Each preganglionic neuron diverges to contact a number of post-ganglionic neurons, on each of which several pre-ganglionic inputs converge. However, only suprathreshold “strong,” or “dominant” synapses are effective in transmitting the CNS signals. Strong synapses differ from the other subthreshold “weak,” or “accessory” inputs: (a) excitatory synaptic currents are >1 nA in their amplitude, (b) 3 to ≈>30 times more quanta of acetylcholine are released, (c) pre-synaptic Ca²⁺ entry through channels resistant to all-known antagonists triggers acetylcholine release, and (d) post-synaptic Ca²⁺ entry boosts and prolongs the nicotinic current. While the majority of postganglionic neurons have only one strong input, a proportion receives two or, rarely, three such inputs. In cells with multiple strong inputs, an equivalent number of discrete Ca²⁺ currents can be evoked at distinct foci electrically distant from the soma, suggesting that each strong input has a unique dendritic association with a cluster of Ca²⁺ channels. When strong preganglionic inputs are destroyed, residual weak synapses sprout and rapidly restore the suprathreshold connections. While much remains to be discovered about how strong synapses are established, their high safety factor ensures the wide and secure distribution of vasoconstrictor command signals from the CNS. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 294–301, July–October, 2007.     

Mg2+ release coupled to Ca2+ uptake: A novel Ca2+ accumulation mechanism in rat liver

Isolated hepatocytes release 2–3 nmol Mg²⁺/mg protein or ~10% of the total cellular Mg²⁺ content within 2 minutes from the addition of agonists that increase cellular cAMP, for example, isoproterenol (ISO). During Mg²⁺ release, a quantitatively similar amount of Ca²⁺ enters the hepatocyte, thus suggesting a stoichiometric exchange ratio of 1 Mg²⁺:1Ca²⁺. Calcium induced Mg²⁺ extrusion is also observed in apical liver plasma membranes (aLPM), in which the process presents the same 1 Mg²⁺:1Ca²⁺ exchange ratio. The uptake of Ca²⁺ for the release of Mg²⁺ occurs in the absence of significant changes in Δψ as evidenced by electroneutral exchange measurements with a tetraphenylphosphonium (TPP⁺) electrode or ³H-TPP⁺. Collapsing the Δψ by high concentrations of TPP⁺ or protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) does not inhibit the Ca²⁺-induced Mg²⁺ extrusion in cells or aLPM. Further, the process is strictly unidirectional, serving only in Ca²⁺ uptake and Mg²⁺ release. These data demonstrate the operation of an electroneutral Ca²⁺/Mg²⁺ exchanger which represents a novel pathway for Ca²⁺ accumulation in liver cells following adrenergic receptor stimulation. This work was supported by National Institutes of Health Grant HL 18708.     

Protein kinase a dependent phosphorylation activates mg2+ efflux in the basolateral region of the liver

Isolated hepatocytes in physiological [Na⁺] ₀ tightly maintain [Mg²⁺] _i . Upon β-adrenergic stimulation or in the presence of permeable cAMP, hepatocytes release 5–10% (1–3 mM Mg²⁺) of their total Mg²⁺ content. However, isolated basolateral liver plasma membranes (bLPM), release Mg²⁺ in the presence of [Na⁺] _o even in the absence of catecholamine stimulation. The data indicate that a physiological brake for Mg²⁺ efflux is present in the hepatocyte and is removed upon cellular signaling. In contrast, this regulation “brake” is absent in purified bLPM thus rendering them fully active. The present study was carried out to reconstruct the missing regulatory component. Activation of Mg²⁺ extrusion in intact cells is consistent with cAMP dependent phosphorylation of the transporter or a regulatory protein. Treatment of bLPM with a non-specific phosphatase such as alkaline phosphatase (AP), decreased Mg²⁺ efflux by 70% compared to untreated bLPM. When AP-treated bLPM were loaded with protein kinase A (PKA), and stimulated with permeable cAMP, Mg²⁺ transport fully recovered. These data suggest that phosphorylation of the Na⁺/Mg²⁺ exchanger or a nearby protein activates the Mg²⁺ transport mechanism in hepatocytes.     

Evaluation of a Two-Site, Three-Barrier Model for Permeation in CaV3.1 (α1G) T-Type Calcium Channels: Ca2+, Ba2+, Mg2+, and Na+

We explored the ability of a two-site, three-barrier (2S3B) Eyring model to describe recently reported data on current flow through open Ca_V3.1 T-type calcium channels, varying Ca²⁺ and Ba²⁺ over a wide range (100 nm–110 mm) while recording whole-cell currents over a wide voltage range (−150 mV to +100 mV) from channels stably expressed in HEK 293 cells. Effects on permeation were isolated using instantaneous current–voltage relationships (IIV) after strong, brief depolarizations to activate channels with minimal inactivation. Most experimental results were reproduced by a 2S3B model. The model described the IIV relationships, apparent affinities for permeation and block for Ca²⁺ and Ba²⁺, and shifts in reversal potential between Ca²⁺ and Ba²⁺. The fit to block by 1 mm  \textMg²⁺_\texti {\text{Mg}}^{2+}_{\text{i}} was reasonable, but block by \textMg²⁺_\texto {\text{Mg}}^{2+}_{\text{o}} was described less well. Surprisingly, fits were comparable with strong ion–ion repulsion, with no repulsion, or with intermediate values. With weak repulsion, there was a single high-affinity site, with a low-affinity site near the cytoplasmic side of the pore. With strong repulsion, the net charge of ions in the pore was near +2 over a relatively wide range of concentration and voltage, suggesting a knockoff mechanism. With strong repulsion, Ba²⁺ preferred the inner site, while Ca²⁺ preferred the outer site, potentially explaining faster entry of Ni²⁺ and other pore blockers when Ba²⁺ is the charge carrier.     

Fifteen-year Change in Forest Floor Organic and Element Content and Cycling at the Turkey Lakes Watershed

To assess the long-term effects of atmospheric deposition on forest floor chemical composition, we took quantitative samplings of L-(Oi), F-(Oe), and H-(Oa) layers at an old-growth sugar maple–yellow birch stand on a till soil at the Turkey Lakes Watershed near Lake Superior, Ontario, Canada, in 1981 and 1996. We then assessed these samples for contents of organic matter (OM), total N, K, Ca, Mg, S, and Na, and exchangeable NH₄ ⁺, NO₃ ⁻, K⁺, Ca²⁺, Mg²⁺, SO₄ ²⁻, and Na⁺. Over the 15-year period, total OM and element contents remained unchanged, with the exception of N, which increased significantly from 61.3 kmol/ha in 1981 to 78.4 kmol/ha in 1996. On an area basis, there were significant increases in exchangeable Ca²⁺ (from 3.8 to 4.6 kmol/ha) and Na⁺ (from 0.05 to 0.08 kmol/ha) and decreases in exchangeable NH₄ ⁺-N (from 1.41 to 0.95 kmol/ha) and SO₄ ²⁻-S (from 1.29 to 0.96 kmol/ha). There were no significant differences in average annual litterfall OM, N, Ca, Mg, S or Na inputs between 1980 and 1985 and between 1992 and 1997. Average annual wet-only SO₄ ²⁻-S deposition during 1981–86 was 0.30; during 1992–97, it was 0.21 kmol/ha. Annual wet-only NO₃ ⁻-N averaged 0.33 kmol/ha during 1981–86 and was similar during 1992–97. Throughfall was less rich in SO₄ ²⁻ and Ca²⁺, Mg²⁺, and Na⁺ during 1992–97 than earlier. Throughfall NH₄ ⁺ and NO₃ ⁻ fluxes were unchanged. Efflux of cations from the forest floor reflected reduced throughput of SO₄ ²⁻. Overall, the results suggest that in spite of atmospheric inputs, active biological processes—including litter input, fine-root turnover, and tree uptake—serve to impart stability to the mineral composition of mature sugar maple forest floor. Received 5 October 1999; accepted 25 October 2000.     

Purification and characterization of a psychrophilic glutathione reductase from Antarctic ice microalgae <Emphasis Type="Italic">Chlamydomonas</Emphasis> sp. Strain ICE-L

A psychrophilic glutathione reductase from Antarctic ice microalgae Chlamydomonas sp. Strain ICE-L was purified by ammonium sulfate fractionation and three steps of chromatography. The yield was up to 25.1% of total glutathione reductase in the crude enzyme extract. The glutathione reductase activity was characterized by the spectrophotometric method under different conditions. Purified glutathione reductase was separated by SDS-PAGE, which furnished a homogeneous band. The native molecular mass of the enzyme was 115 kDa. Apparent Km values for NADPH and NADH (both at 0.5 mmol L⁻¹ oxidized glutathione) were 22.3 and 83.8 μmol L⁻¹, respectively. It was optimally active at pH 7.5, and it was stable from pH 5 to 9. Its optimum temperature was 25°C, with activity at 0°C 23.5% of the maximum. Its optimum ion strength and optimum Mg²⁺ were 50–90 and 7.5 mmol L⁻¹, respectively. Ca²⁺, Mg²⁺, and cysteine substantially increased the activity of the enzyme but chelating agents, heavy metals (Cd²⁺, Pb²⁺, Cu²⁺, Zn²⁺, etc.), NADPH, and ADP had significant inhibitory effects. This glutathione reductase can be used to study the adaptation and mechanism of catalysis of psychrophilic enzymes, and it has a high potential as an environmental biochemical indicator under extreme conditions.     

Mg2+/Ca2+-ATPase Activity Is Not Enriched in Synaptic Vesicles Isolated from Rat Cerebral Cortex

Neuronal ATPases comprise a wide variety of enzymes which are not uniformly distributed in different membrane preparations. Since purified vesicle fractions have Mg²⁺/Ca²⁺-ATPase, the purpose of the present study was to know whether such enzyme activities have a preferential concentration in a synaptic vesicle fraction in order to be used as markers for these organelles. Resorting to a procedure developed in this Institute, we fractionated the rat cerebral cortex by differential centrifugation following osmotic shock of a crude mitochondrial fraction and separated a purified synaptic vesicle fraction over discontinuous sucrose gradients. Mg²⁺/Ca²⁺-ATPase activities and ultrastructural studies of isolated fractions were carried out. It was observed that similar specific activities for Mg²⁺/Ca²⁺-ATPases were found in all fractions studied which contain synaptic vesicles and/or membranes. Although the present results confirm the presence of Mg²⁺ and Ca²⁺-ATPase activities in synaptic vesicles preparations, they do not favor the contention that Mg²⁺/Ca²⁺-ATPase is a good marker for synaptic vesicles.     

Investigation on the micro-mechanisms of Al3+ interfering the reactivities of aspartic acid and its biological processes with Mg2+

Density functional theory (DFT) has been applied to study the micro-mechanisms of Al³⁺ interfering the reactivities of aspartic acid (H₂asp) and its biological processes with Mg²⁺. All the 46 stable conformers of Hasp^- and 3 of asp²⁻ have been determined at the B3LYP/6-311++G** level, showing that the 7 most stable conformers of Hasp⁻ all present a very strong and linear O–H···O H-bond between carboxyl and carboxylic acid groups with the bond energy high up to 162 kJ mol⁻¹. The reaction thermodynamics and micro-mechanism between Al³⁺ and Hasp⁻ (or asp²⁻) in aqueous phase have been investigated by the combined application of supramolecular model and polarizable continuum IEFPCM solvent model, firstly revealing Al³⁺ interfering in the biological processes of aspartic acid. The substitution thermodynamics and mechanisms of Mg²⁺ by Al³⁺ in the biological processes between the species of aspartic acid and Mg²⁺ in aqueous phase were probed, revealing the facile displacement of Mg²⁺ by Al³⁺. These results may provide a reasonable mechanism of Al³⁺ biological toxicity at the microscopic level.     

Synaptosomal non-mitochondrial ATPase activities and drug treatment

Energy-using non-mitochondrial ATPases were assayed in rat cerebral cortex synaptosomes and synaptosomal subfractions, namely synaptosomal plasma membranes and synaptic vesicles. The following enzyme activities were evaluated: Na⁺, K⁺-ATPase; high- and low-affinity Ca²⁺-ATPase; basal Mg²⁺-ATPase; Ca²⁺, Mg²⁺-ATPase. The evaluations were performed after four week-treatment with saline [controls] or -adrenergic agents (-yohimbine, clonidine), energymetabolism interfering compound (theniloxazine), and oxygen-partial pressure increasing agent (almitrine), in order to define the plasticity and the selective changes in individual ATPases. In rat cerebral cortex, the enzyme adaptation to four-week-treatment with -yohimbine or clonidine was characterized by increase in both high- and low-affinity Ca²⁺-ATPase activities. The action involves the enzyme form located in the synaptic plasma membranes. The enzyme adaptation to the subchronic treatments with theniloxazine or almitrine was characterized by increase in Na⁺, K⁺-ATPase or Mg²⁺-ATPase activities, respectively. The action involves the enzymatic forms located in the synaptic plasma membranes. Thus, the pharmacodynamic effects of the agents tested should also be related to the changes induced in the activity of some specific synaptosomal nonmitochondrial ATPases.     

Effect of In Vivo Administration of Naloxone on ATP-ase's Enzyme Systems of Synaptic Plasma Membranes from Rat Cerebral Cortex

Naloxone is a specific competitive antagonist of morphine, acting on opiate receptors, located on neuronal membranes. The effects of in vivo administration of naloxone on energy-consuming non-mitochondrial ATP-ases were studied in two different types of synaptic plasma membranes from rat cerebral cortex, known to contain a high density of opiate receptors. The enzyme activities of Na⁺, K⁺-ATP-ase, Ca²⁺, Mg²⁺-ATP-ase and Mg²⁺-ATP-ase and of acetylcholinesterase (AChE) were evaluated on synaptic plasma membranes obtained from control and treated animals with effective dose of naloxone (12g · kg^–1 i.m. 30 minutes). In control (vehicle-treated) animals specific enzyme activities assayed on these two types of synaptic plasma membranes are different, being higher on synaptic plasma membranes of II type than of I type, because the first fraction is more enriched in synaptic plasma membranes. The acute treatment with naloxone produced a significant decrease in Ca²⁺,Mg²⁺-ATP-ase activity and an increase in AChE activity, only in synaptic plasma membranes of II type. The decrease of Ca²⁺,Mg²⁺-ATP-ase enzymatic activity and the increased AChE activity are related to the interference of the drug on Ca²⁺ homeostasis in synaptosoplasm, that leads to the activation of calcium-dependent processes, i.e. the extrusion of neurotransmitter. These findings give further evidence that pharmacodynamic characteristics of naloxone are also related to increase [Ca²⁺] _i , interfering with enzyme systems (Ca²⁺,Mg²⁺-ATP-ase) and that this drug increases acetylcholine catabolism in synaptic plasma membranes of cerebral cortex.     

Effect of B1 hypovitaminosis on the efficacy of neuromuscular transmission in the murine diaphragm

In mice kept on a diet with no vitamin B₁, the total content of thiamine in the brain decreased, and muscle contractions evoked by stimulation of the nerve in phrenico-diaphragmatic preparations obtained from such animals became weaker. The measurements were performed in Krebs solution with a decreased content of Ca²⁺ and increased concentration of Mg²⁺; values of the developed force were normalized with respect to those in normal Krebs solution. Thus, B₁ hypovitaminosis results in a decrease in the efficacy of neuromuscular synaptic transmission. Some neurological symptoms typical of a deficiency of vitamin B₁ in the organism can be related to this effect. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 416–418, July–October, 2007.     

Magnesium Inhibition of Ryanodine-Receptor Calcium Channels: Evidence for Two Independent Mechanisms

The gating of ryanodine receptor calcium release channels (RyRs) depends on myoplasmic Ca²⁺ and Mg²⁺ concentrations. RyRs from skeletal and cardiac muscle are activated by μm Ca²⁺ and inhibited by mm Ca²⁺ and Mg²⁺. ⁴⁵Ca²⁺ release from skeletal SR vesicles suggests two mechanisms for Mg²⁺-inhibition (Meissner, Darling & Eveleth, 1986, Biochemistry 25:236–244). The present study investigates the nature of these mechanisms using measurements of single-channel activity from cardiac- and skeletal RyRs incorporated into planar lipid bilayers. Our measurements of Mg²⁺- and Ca²⁺-dependent gating kinetics confirm that there are two mechanisms for Mg²⁺ inhibition (Type I and II inhibition) in skeletal and cardiac RyRs. The mechanisms operate concurrently, are independent and are associated with different parts of the channel protein. Mg²⁺ reduces P _o by competing with Ca²⁺ for the activation site (Type-I) or binding to more than one, and probably two low affinity inhibition sites which do not discriminate between Ca²⁺ and Mg²⁺ (Type-II). The relative contributions of the two inhibition mechanisms to the total Mg²⁺ effect depend on cytoplasmic [Ca²⁺] in such a way that Mg²⁺ inhibition has the properties of Types-I and II inhibition at low and high [Ca²⁺] respectively. Both mechanisms are equally important when [Ca²⁺] = 10 μm in cardiac RyRs or 1 μm in skeletal RyRs. We show that Type-I inhibition is not the sole mechanism responsible for Mg²⁺ inhibition, as is often assumed, and we discuss the physiological implications of this finding. Received: 1 January 1996/Revised: 14 November 1996     

Effect of Mg2+ on the Structure and Function of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase

Mg²⁺ in various concentrations was added to purified Rubisco in vitro to gain insight into the mechanism of molecular interactions between Mg²⁺ and Rubisco. The enzyme activity assays showed that the reaction between Rubisco and Mg²⁺ was two order, which means that the enhancement of Rubisco activity was accelerated by low concentration of Mg²⁺ and slowed by high concentration of Mg²⁺. The kinetics constant (K _m) and V _max was 1.91 μM and 1.13 μmol CO₂ mg⁻¹ protein∙min⁻¹, respectively, at a low concentration of Mg²⁺, and 3.45 μM and 0.32 μmol CO₂∙mg⁻¹ protein∙min⁻¹, respectively, at a high concentration of Mg²⁺. By UV absorption and fluorescence spectroscopy assays, the Mg²⁺ was determined to be directly bound to Rubisco; the binding site of Mg²⁺ to Rubisco was 0.275, the binding constants (K _A) of the binding site were 6.33 × 10⁴ and 5.5 × 10⁴ l·mol⁻¹. Based on the analysis of the circular dichroism (CD) spectra, it was concluded that the binding of Mg²⁺ did not alter the secondary structure of Rubisco, suggesting that the observed enhancement of Rubisco carboxylase activity was caused by a subtle structural change in the active site through the formation of the complex with Mg²⁺.