Action of tetanus toxin and colchicine on synaptic membranes of the rat cerebral cortex

Purified tetanus toxin (TT), in experiments in vitro, was shown to affect neither the Na,K-ATPase activity of the synaptic membrane fraction of the rat cerebral cortex nor the inhibition of Na,K-ATPase activity produced by electrical stimulation of a suspension of synaptic membranes, nor the binding of GABA-³H by synaptosomes. TT and colchicine (1 mM) reduced the osmotic sensitivity of the nerve endings. Colchicine, in low concentrations (10^–5 to 10^–3 M), does not affect Mg- and Na,K-ATPase but, in higher concentrations (10^–2 M), it inhibits the activity of both ATPases considerably.Laboratory of General Pathology of the Nervous System, Institute of General Pathology and Pathological Physiology, Academy of Sciences of the USSR, Moscow. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 2, pp. 139–142, February, 1977.     

Effect of benactyzine and arecoline on Mg2–-ATPase activity and content of Ca2– and Mg2– ions in the rat brain

The effect of the central cholinolytic benactyzine (40 mg/kg) and of the cholinomimetic arecoline (2.5 mg/kg) on activity of Mg^2–-dependent ATPase was studied and the content of Ca^2– and Mg^2– ions determined in rat brain. Benactyzine and arecoline caused biphasic changes in the activity of the enzyme and content of the electrolytes. It is concluded that inhibition of the enzyme is linked with the accumulation of Ca^2– ions and its activation with an increase in the concentration of Mg^2– ions in brain tissue. It is suggested that benactyzine and arecoline exert their influence on the liberation and retention of neuromediators in the tissue depots through these effects.(Presented by Academician of the Academy of Medical Sciences of the USSR V. V. Zakusov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol.84, No. 9, pp. 306–309, September, 1977.     

Comparative study of the effect of amiridine on biological membranes

The effects of amiridine and of the comparable drugs tacrine and piracetam on synaptosomes and membranes of sarcoplasmic reticulum were studied by electron paramagnetic resonance; in addition, the effects of these drugs on the activity of Ca²⁺, Mg²⁺-dependent ATPase regulating calcium transport in neurons were investigated. In concentrations of 10⁻⁷ to 10⁻⁵ M the drugs did not affect the structure of synaptosomal membranes of rat brain. Amiridine and tacrine in a concentration of 0.1 mM reduced the rate of calcium ion transport across the sarcoplasmic reticulum membrane by inhibiting the function of Ca²⁺, Mg²⁺-dependent ATPase and induced marked changes of the structural rigidity of the protein part of the membrane. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 120, N№ 11, pp. 503–505, November, 1995 Presented by Yu. A. Romanov, Member of the Russian Academy of Medical Sciences     

Sensitivity of RNA-synthesizing system of the lymphocytes of patients with malignant neoplasms to phytohemagglutinin and dexamethasone

Biosynthesis of RNA catalyzed by DNA-dependent RNA polymerase was demonstrated in a reconstructed system containing isolated lymphocyte nuclei, Mg²⁺ or Mn²⁺ salts, and ammonium sulfate, in the presence of four nucleoside triphosphates. Both Mg²⁺- and Mn²⁺-dependent forms of this enzyme were found in the nuclei of normal lymphocytes and of lymphocytes from patients with melanoma, lung carcinoma, and sarcoma. The activity of both forms of RNA polymerase in the nuclei of the patients' lymphocytes was higher than in normal analogs. The sensitivity of DNA-dependent RNA polymerase to dexamethasone and phytohemagglutinin was less marked in the nuclei from patients with lung carcinoma, melanoma, and sarcoma than in normal lymphocytes.Laboratory of Biochemistry of Tumors, All-Union Oncologic Center of the USSR, Moscow. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 4, pp. 449–452, April, 1977.     

Effect of activation of Ca2+, Mg2+-dependent endonuclease on proliferative response of human peripheral blood lymphocytes

Activity of Ca²⁺, Mg²⁺-dependent endonuclease in human peripheral blood mononuclears is studied. Intact cells exhibit extremely low activity of the enzyme. Treatment with the synthetic hexapeptide imunofan considerably stimulates endonuclease activity in mononuclears. This activation does not depend on additional protein synthesis. An 1-h incubation in the presence of cycloheximide also activates Ca²⁺, Mg²⁺-dependent endonuclease. These data suggest that imunofant and/or cycloheximide activate the apoptotic cascade. This leads to activation of endonuclease, which is not synthesizedde novo but persists in the cell in the form of inactive precursor. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 123, No. 5, pp. 535–537, May, 1997     

Initiation of apoptosis in hepatocytes during prolonged arterial hypotension and postresuscitation period

An increase in activity of Ca²⁺, Mg²⁺-dependent endonucleases on the second hour of hypotension coincided with the presence of DNA fragments in agarose gel. A correlation was established between the duration of hypotension, Ca²⁺, Mg²⁺-dependent endonuclease activity, and intensity of nuclear DNA fragmention. Apopotosis of hepatocytes is triggered during ischemia and develops during reperfusion. Translated fromByulleten' Eksperimental'noi Biologii i Meditisiny, Vol. 125, No. 3, pp. 285–288, March, 1998     

Type B monoamine oxidase and functions of Ca2+, Mg2+-dependent adenosinetriphosphatase in preparations from sarcoplasmic reticulum vesicles

Oxidative deamination of -phenylethylamine or benzylamine by type B monoamine oxidases (MAO) in preparations of sarcoplasmic reticulum vesicles from rabbit skeletal muscles is accompanied by inhibition both of active Ca²⁺ transport into the vesicles and of the activity of Ca²⁺, Mg²⁺-dependent ATPase, which is preventable by deprenil, a specific inhibitor of type B MAO. Aldehydes formed during enzymatic deamination of substrates of type B MAO may perhaps participate in the regulation of Ca²⁺, Mg²⁺-dependent ATPase, activity.Laboratory of Physicochemical Methods, Scientific-Research Institute for Biological Trials of Chemical Compounds, Ministry of the Medical Industry of the USSR. Laboratory or Biochemistry of Amines and Other Nitrogenous Compounds, Institute of Biological and Medical Chemistry, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR V. N. Orekhovich.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 3, pp. 283–284, March, 1977.     

Na+, K+-ATPase activity in neurons and glial cells of the olfactory cortex of the rat brain during the development of long-term potentiation

Na⁺, K⁺-ATPase and Mg²⁺-ATPase activities were studied in neurons and glial cells of the olfactory cortex of the rat by quantitative cytophotometry in conditions of long-term potentiation (LTP), and significant changes in direction and extent were found. Na⁺, K⁺-ATPase activity decreased in neurons in the first 15 min after LTP, with subsequent elevation by 30 min. Mg²⁺-ATPase activity remained unchanged in these conditions. Glial cells showed significant increases in Na⁺, K⁺-ATPase activity in the initial period after LTP, with return to control by 30 min. Again, there were no significant changes in Mg²⁺-ATPase activity. The formation and persistence of LTP in neurons and glial cells was accompanied by significant changes in Na⁺, K⁺-ATPase activity, which were reciprocal in nature. Functional Neurochemistry Laboratory (Director N. A. Emel'yanov), I. P. Pavlov Institute of Physiology, Russian Academy of Sciences, St. Petersburg. Translated from Fiziologicheskii Zhurnal im. I. M. Sechenova, Vol. 81, No. 3, pp. 16–20, March, 1995.     

Investigation of the mechanism of action of ouabain and cyclic AMP of the transport of calcium ions by rat heart mitochondria

The action of ouabain and cyclic AMP on the Ca-accumulating capacity and outflow of Ca²⁺ ions from loaded rat heart mitochondria was studied by the tetracycline probe method. In the course of the investigations no effect of ouabain on these processes was found. Cyclic AMP did not act on Ca binding by the mitochondrial membrane but it induced rapid liberation of Ca²⁺ from organelles loaded with these ions.Department of Molecular Pharmacology, Medico-Biological Faculty, N. I. Pirogov Second Moscow Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR S. S. debov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 2, pp. 158–160, February, 1977.     

Inhibition of ω-conotoxin-sensitive Ca2+ channel currents by internal Mg2+ in cultured rat cerebellar granule neurones

The effects of changing the intracellular concentrations of either free Mg²⁺ ions ([Mg²⁺]_i) or Mg²⁺-bound adenosine triphosphate ([Mg · ATP]_i) on Ca²⁺ channel currents were assessed in cultured rat cerebellar granule neurones using the whole-cell patch-clamp technique. Raising [Mg²⁺]_i from 0.06 mM to 1.0 mM inhibited Ca²⁺ channel currents by approximately 50%. The action of -conotoxin GVIA (-CgTX), a selective inhibitor of N-type Ca²⁺ channels was also investigated. With increasing [Mg²⁺]_i, the proportion of current irreversibly blocked by -CgTX was reduced, and was negligible (approximately 5 pA of current) in the presence of [Mg²⁺]_i values of 0.5 mM or greater. Block of the -CgTX-sensitive current accounted for the reduction in total current by concentrations of [Mg²⁺]_i to 0.5 mM. Raising [Mg²⁺]_i had no effect on the rate of decay of Ca²⁺ currents, but did produce a negative shift in current activation, possibly due to a non-specific interaction with negative surface charge. Altering [Mg · ATP]_i from 0.3 to 5.0 mM caused a twofold increase in the size of currents without affecting the proportion of current sensitive to -CgTX. [Mg²⁺]_i was also effective in inhibiting the Ca²⁺ channel current following potentiation by increasing [Mg · ATP]_i. These data suggest that -CgTX-sensitive current in these cells is selectively inhibited by internal Mg²⁺ whereas both -CgTX-sensitive and -resistant components of current are potentiated by internal Mg · ATP. The mechanism by which Mg²⁺ inhibits N-type channels is unclear, but may involve an open channel block.     

Effect of L-arginine-NO system on the erythrocyte ATPase activity and LPO

Activity of Na,K-ATPase in erythrocytes of children with asthmatic bronchitis and cerebral palsy rapidly changed under the effect of NO synthesis inhibitor L-NAME and did not depend on the plasma Ca²⁺ concentration. Enzyme activities correlated with the content of Mg²⁺; and a tendency toward accumulation of LPO substrates and primary molecular LPO products was noted. The relative content of Schiff bases increased almost 2-fold. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 128, No. 9, pp. 321–323, September, 1999     

Effects of forskolin on crypt cells of rat distal colon

Na⁺-K⁺-2Cl^– cotransport activity has previously been shown to depend on both intracellular ATP and Mg²⁺, but the mechanisms remain unknown. Cotransport in avian erythrocytes can be stimulated by a variety of agents including cAMP and permeant serine/ threonine phosphatase inhibitors and is inhibited by prior depletion of either ATP with antimycin A, or Mg²⁺ by incubation in A23187 plus EDTA. However, when cells were first stimulated with cAMP rather than calyculin A then subjected to either depletion strategy, a differential effect was found. The phosphatase-inhibitor-treated cells were resistant to subsequent ATP or Mg²⁺ depletion while cAMP-treated cells were sensitive to both treatments. Parallel examination of protein phosphorylation confirmed that ATP or Mg²⁺ depletion leads to dephosphorylation of membrane proteins in cAMP-treated but not in calyculin-A-treated cells. These results suggest that, for cotransport, ATP and Mg²⁺ are required primarily to maintain the system in a phosphorylated state rather than as direct modulators. The relative effectiveness of okadaic acid (EC₅₀ 630 nM) and calyculin A (EC₅₀ 8 nM) in stimulating the cotransporter indicate that a type-1 protein phosphatase is probably responsible for dephosphorylating the system. Cells stimulated by hypertonicity were also resistant to ATP or Mg²⁺ depletion suggesting that the mechanism of shrinkage-induced cotransport stimulation might also involve protein phosphatase modulation.     

Human Kir2.1 channel carries a transient outward potassium current with inward rectification

We have previously reported a depolarization-activated 4-aminopyridine-resistant transient outward K⁺ current with inward rectification (I _to.ir) in canine and guinea pig cardiac myocytes. However, molecular identity of this current is not clear. The present study was designed to investigate whether Kir2.1 channel carries this current in stably transfected human embryonic kidney (HEK) 293 cells using whole-cell patch-clamp technique. It was found that HEK 293 cells stably expressing human Kir2.1 gene had a transient outward current elicited by voltage steps positive to the membrane potential (around −70 mV). The current exhibited a current–voltage relationship with intermediate inward rectification and showed time-dependent inactivation and rapid recovery from inactivation. The half potential (V _0.5) of availability of the current was −49.4 ± 2.1 mV at 5 mM K⁺ in bath solution. Action potential waveform clamp revealed two components of outward currents; one was immediately elicited and then rapidly inactivated during depolarization, and another was slowly activated during repolarization of action potential. These properties were similar to those of I _to.ir observed previously in native cardiac myocytes. Interestingly, inactivation of the I _to.ir was strongly slowed by increasing intracellular free Mg²⁺ (Mg²⁺ _i , from 0.03 to 1.0, 4.0, and 8.0 mM). The component elicited by action potential depolarization increased with the elevation of Mg²⁺ _i . Inclusion of spermine (100 μM) in the pipette solution remarkably inhibited both the I _to.ir and steady-state current. These results demonstrate that the Mg²⁺ _i -dependent current carried by Kir2.1 likely is the molecular identity of I _to.ir observed previously in cardiac myocytes.     

Intracellular Mg2+ inhibits the IP3-activated I K(Ca) in NG108-15 cells. [Why intracellular citrate can be useful for recording I K(Ca)]

Receptor-mediated formation of inositol 1,4,5-trisphosphate (IP₃) can induce an outward Ca²⁺-activated K⁺ current [I _K(Ca)] some neural cells. We have investigated I _K(Ca) activated by intracellular injections of IP₃ in whole-cell patch-clamped neuroblastoma×glioma hybrid cells. The current could only be recorded reliably using citrate as the anion in the pipette, but not using acetate, aspartate, chloride, fluoride, gluconate or methylsulphate. This could be attributed to buffering of intracellular Mg²⁺ by citrate. Theoretical calculations suggested free [Mg²⁺] of 1.0 and 0.07 mM respectively in the acetate- and citrate-based recording solutions. Further, IP₃-activated I _K(Ca) could be recorded when the free Mg²⁺ level in the acetate, chloride or methylsulphate solutions was lowered to the range (0.05 mM) calculated for the citrate solution. Thus, raised [Mg²⁺] blocks I _K(Ca). This appeared to be due to inhibition of the response to released Ca²⁺, since high [Mg²⁺] also blocked the response to intracellular injections of Ca²⁺ ions. Mean Mg²⁺ levels in intact neuroblastoma×glioma hybrid cells measured by Mag-Indo-1/AM fluorescence were estimated to be less than 0.14 mM. We therefore conclude that IP₃-induced I _K(Ca) is expressed under normal conditions, but may be subject to regulation by intracellular Mg²⁺.     

Effect of tetraethylammonium on electrophysiological properties of smooth-muscle cells of the pulmonary artery

Electrical properties of single muscle cells were investigated by a microelectrode method and the contractile activity of the pulmonary artery was studied during the action of tetraethylammonium (TEA). Smooth-muscle cells in the presence of TEA were shown to be able to generate both spontaneous action potentials (APs) and APs evoked by stimulation of these cells by a depolarizing current. The appearance of the AP must evidently be connected with depression by TEA of the delayed potential-dependent potassium current, early activation of which in normal solution prevents AP development. APs in muscle cells are generated by the entry of Ca²⁺ ions into the cell and they are therefore blocked by Mn²⁺ ions. Besides the specific action of TEA, additional effects of it also are observed: a) its depolarizing action, accompanied by an increase in membrane resistance; b) stimulation of the entry of Ca²⁺ ions into muscle cells from the external medium, evoking tonic contraction of the muscle; c) the formation of a connection between the resting potential and contraction.Department of Neuromuscular Physiology, A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. (Presented by Academician of the Academy of Medical Sciences of the USSR N. N. Gorev.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 2, pp. 134–136, February, 1977.     

Intracellular free Mg2+ concentration in skeletal muscle fibres of frog and crayfish

The free intracellular Mg²⁺ concentration ([Mg²⁺]_i) was investigated in frog sartorius and crayfish phasic and tonic skeletal muscle fibres, using a new Mg²⁺-sensitive microelectrode based on the ionophore ETH 5214 [Hu et al. (1989) Anal Chem 61:574–576]. In Ringer solution containing 0.5 mmol/l MgCl₂, the mean [Mg²⁺]_i of the frog muscle fibres was 1.3 mmol/l. In phasic crayfish muscle fibres, [Mg²⁺]_i was about twice as high (mean 3.5 mmol/l) as in tonic fibres (mean 1.5 mmol/l), measured in van Harreveld solution containing 1.2 mmol/l MgCl₂. Long-lasting (3–12 h) incubation of frog skeletal muscle fibres in Na⁺-free solution produced a reversible increase of [Mg²⁺]_i by a factor of about 1.7. A tenfold rise of extracellular Mg²⁺ led to an increase in [Mg²⁺]_i in the presence as well as in the absence of Na⁺. In these experiments, mean [Mg²⁺]_i values of 3.2 mmol/l were never exceeded. Thus, [Mg²⁺]_i remained at least 60 times lower than predicted from a passive distribution across the cell membrane. The results suggest the existence of a Na⁺-dependent and a Na⁺-independent Mg²⁺ extrusion mechanism, which is regulated by actual Mg²⁺ concentrations.     

Characteristics of ecto-ATPase of Xenopus oocytes and the inhibitory actions of suramin on ATP breakdown

Ecto-ATPase activity of Xenopus oocytes was studied by measuring the production of inorganic phosphate (P_i) from the breakdown of extracellular ATP. Enzyme activity involved Ca²⁺/Mg²⁺-dependent and Ca²⁺/Mg²⁺-independent dephosphorylation of ATP. Ca²⁺/Mg²⁺-dependent ecto-ATPase was active over a limited range of 0.01–1.0 mM ATP, while Ca²⁺/Mg²⁺-independent ATPase activity was active over a range of 0.1–30 mM ATP. Total enzyme activity was insensitive to changes in buffer pH (pH 7.0–9.0), but increased in a relatively linear manner with: (1) time of reaction (0–90 min), (2) number of cells (1–20 oocytes), and (3) temperature (10–37°C). Ecto-ATPase activity was unaffected by ouabain (100 M), sodium azide (100 M), and oligomycin (5 g/ml) (as inhibitors of endo-ATPases) and -glycerophosphate (10 mM) and p-nitrophenyl phosphate (10 mM) (as inhibitors of non-specific alkaline phosphatase). Total ecto-ATPase activity was reduced significantly in defolliculated oocytes, suggesting that the enzyme was located mainly on the enveloping follicle cell layer. The range order of preferential substrates was: ATP>GTP, ITP, UTP, CTP, TTP, 2-methylthioATP>ADP, 2-methylthioADP, AMP,-methylene ATP, ,-methylene ATP, in the presence of divalent ions (where G is guanosine, I is inosine, U is uridine, C is cytidine and T is ribosylthymine). The P₂-purinoceptor antagonist suramin [8-(3-benzamido-4-methylbenzamido) napthalene-1,3,5-trisulphonic acid), 100 M] significantly inhibited total ecto-ATPase activity; this inhibition was competitive for the Ca²⁺/Mg²⁺-dependent enzyme. This striking property of suramin may point to a structural similarity between the ATP-binding sites of ecto-ATPase and purinoceptors, a potentially complicating factor where purinoceptors expressed in oocytes are used to test the potency of agonists and the efficacy of receptor antagonists and enzyme inhibitors.     

Ultraviolet spectrophotometric studies of the reactivity of a water-soluble polymer containing pendant 8-hydroxyquinoline moieties with metal ions

The water-soluble polymer 4 containing pendant 8-hydroxyquinoline (oxine) moieties was prepared and its reactivity with metal ions was studied by means of ultraviolet (UV) spectrophotometry. Polymer 4 was found to form complexes with metal ions in a mole ratio of 2 : 1 (oxine : metal), which is also obtained for the metal complexes of model compound 3 except for the Mg²⁺ complex. Polymer 4 hardly reacts with Mg²⁺, while model compound 3 forms a 1 : 1 mol/mol complex. The stability of the complexes of polymer 4 with metal ions was found to decrease in the following order: Cu²⁺ > Ni²⁺ > Zn²⁺ > Cd²⁺ > Hg²⁺ > Mg²⁺.     

Influence of extracellur Ca2+ on endogenous Cl− channels in Xenopus oocytes

Removal of Ca²⁺ from the external bath solution evoked marked depolarization and large currents (up to several microamperes) in voltage-clamped defolliculated oocytes of Xenopus laevis. The resulting current was not carried by a cation influx but was due to a huge Cl^– efflux, which could be strongly inhibited by the Cl^– channel blockers flufenamic acid and niflumic acid. Removal of Mg²⁺ or Ba²⁺ from the solutions had the same effects as removing Ca²⁺. The reversal potential of –12 mV also indicated that Cl^– channels were responsible for the large currents. Patch-clamp studies revealed a single-channel slope conductance of 90 pS. During oocyte maturation these channels remained active. The half-maximal Ca²⁺ concentration of about 20 M showed that quite low doses of extracellular Ca²⁺ profoundly influence the electrical properties of the oocyte membrane.     

A Mg-dependent ecto-ATPase is increased in the infective stages of<Emphasis Type="Italic"> Trypanosoma cruzi</Emphasis>

In this work, we describe the ability of living epimastigotes of Trypanosoma cruzi to hydrolyze extracellular ATP. In these intact parasites, there was a low level of ATP hydrolysis in the absence of any divalent metal (2.42±0.31 nmol Pi/h×10⁸ cells). ATP hydrolysis was stimulated by MgCl₂, and the Mg-dependent ecto-ATPase activity was 27.15±2.91 nmol Pi/h×10⁸ cells. The addition of MgCl₂ to the extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. This stimulatory activity was also observed when MgCl₂ was replaced by MnCl₂, but not by CaCl₂ or SrCl₂. The apparent K_m for Mg-ATP^2– was 0.61 mM, and free Mg²⁺ did not increase the ecto-ATPase activity. This ecto-ATPase activity was insensitive to the inhibitors of other ATPase and phosphatase activities. To confirm that this Mg-dependent ATPase was an ecto-ATPase, we used an impermeant inhibitor, DIDS (4, 4.diisothiocyanostylbene 2-2-disulfonic acid) as well as suramin, an antagonist of P₂ purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg²⁺-dependent ATPase activity in a dose-dependent manner. A comparison among the Mg²⁺-ecto-ATPase activities of the three forms of T. cruzi showed that the noninfective epimastigotes were less efficient at hydrolyzing ATP than the infective trypomastigote and amastigote stages.