Acetylcholinesterase activity in anAedes aegypti cell line

Summary Considerable acetylcholinesterase (AChE) activity was detected in anAedes aegypti established cell line. The enzyme is blocked by 10^–6 M eserine sulfate, displays excess substrate inhibition and slowly hydrolyzes butyrylthiocholine. A 2-fold stimulation of AChE activity was shown after 2 days exposure to 3×10^–7 M -ecdysone. AChE activity found in the fresh medium is the contribution of the fetal calf serum portion. A direct relationship between levels of serum and the AChE activity in the cultured cells was demonstrated.Acknowledgment. I wish to thank Dr J. Peleg of the Israel Institute for Biological Research for providing the starting culture ofAedes aegypti cells.     

‘Binding’ of glycine andγ-aminobutyric acid to synaptosomal fractions of 6 regions of the feline brain; effects of strychnine

Summary GABA (6×10^–6 M) binding to synaptosome-enriched fractions of cat CNS exhibited a clear rostro-caudal gradient, whereas glycine (6×10^–6 M) binding was greatest to particles of cerebellar cortex, and this was followed by medulla caudate nucleus cerebral cortex pons > corona radiata. Strychnine-SO₄ (10^–3 or 10^–4 M) inhibited the binding of GABA and glycine in all brain regions studied; at 10^–5 M this drug inhibited the binding of both GABA and glycine only to particles of the cerebral cortex.This study was supported by Centro Nacional Ramón y Cajal and Fundación Juan March. P. M. was a summer student from Eastern Nazarene College, Wollaston, Mass., USA.     

Inhibition of respiratory oxidation of reduced sulfur compounds by intact cells ofThiobacillus denitrificans (strain RT) grown on thiosulfate

Thiobacillus denitrificans strain RT, an obligate sulfur-oxidizing chemolithoautotroph, was grown under microaerophilic conditions with thiosulfate as the only energy source. The rates of tetrathionate, thiosulfate, elemental sulfur (S^o) and sulfite oxidation were measured respirometrically with an oxygen electrode, using actively growing cells. Cells oxidized thiosulfate, elemental sulfur (S^o) and sulfite, but not tetrathionate. The thiosulfateoxidizing activity and elemental sulfur-oxidizing activity (SOA) were almost totally inhibited by 50 M myxothiazol (>80%), an inhibitor of the quinone-cytochrome b region, and by 10 M of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) (>82%). Sulfite-oxidizing activity was also significantly inhibited (>60%) by 50 M myxothiazol and 10 M CCCP. 1 mM KCN totally inhibited (>90%) all respiratory activities. This study confirms that a sulfur-oxidizing activity appears during microaerophilic growth ofThiobacillus denitrificans strain RT on thiosulfate. The SOA is linked to the respiratory chain, probably releasing electrons in the quinone-cytochrome b region.To whom correspondence should be addressed. Submitted by R. Bachofen.     

Evidence against a role of (Na++K+-ATPase in the alpha-adrenoceptor mediated positive inotropic effect of phenylephrine

Summary Phenylephrine (0.1–100 M) in the presence of 1 M propranolol increased the force of contraction in electrically driven papillary muscles from cats. This presumably alpha-adrenoceptor mediated positive inotropic effect of phenylephrine occurred without any influence on (Na⁺+K⁺-ATPase activity.This work was supported by the Deutsche Forschungsgemeinschaft.     

Effects of cerebral lateral,ventricular infusions of phloridzin on feeding and body weight in Gallus domesticus (L.)

Summary Contrary to earlier findings in rats, cerebral lateral ventricular infusions of 1×10^–3 M or 2×10^–3 M solutions of phloridzin at a rate of 2.5 l/min for 90 min had no significant stimulating effects on food intake and weight gain in hens and cocks. These different responses to intraventricular phloridzin might reflect a difference of sensitivity to the inhibitory action of phloridzin on glucose transport in cerebral cells or certain peculiarities of mechanisms controlling food intake in chickens.     

Heterogeneous responses of canine basilar and middle cerebral arteries to serotonin at normal and high CO2 tension

The responses of basilar arteries (BAs) to serotonin were attenuated by high \(P_{CO_2 } \) (86±1 mm Hg) and the pH matched acidotic solution ( \(P_{CO_2 } \) 37±1 mm Hg), whereas the responses of middle cerebral arteries (MCAs) were not. High \(P_{CO_2 } \) decreased the basal tone of both arteries, and the changes in basal tone due to high \(P_{CO_2 } \) were not influenced by 3×10^?7 M imipramine, 10^?5 M pargyline or 10^?4 M aspirin. The responses of BAs to serotonin were attenuated by high \(P_{CO_2 } \) in the presence of imipramine, pargyline and aspirin. The responses of MCAs to serotonin were not influenced by high \(P_{CO_2 } \) in the presence of pargyline and aspirin, but attenuated by high \(P_{CO_2 } \) in the presence of imipramine.     

Inhibition of hepatic Na+, K+-adenosinetriphosphatase in taurolithocholate-induced cholestasis in the rat

Summary Na⁺, K⁺-adenosinetriphosphatase (Na⁺, K⁺-ATPase) activity was decreased in liver plasma membranes from rats in which cholestasis had been induced by i.v. administration of sodium taurolithocholate (5 moles/100 g b. wt). Incubation of liver plasma membranes with taurolithocholate (10–1300 M) caused significant and dose dependent reductions of Na⁺, K⁺-ATPase activity at taurolithocholate concentrations above 100 M. These findings lend support to the hypothesis that cholestasis induced by monohydroxy bile acids is at least partially the result of an inhibition of hepatic Na⁺, K⁺-ATPase activity.This work was supported by the Swiss National Science Foundation.The authors thank Mr H. Sägesser and Miss B. Schütz for technical assistance.     

Effects of prostaglandin E1 on45Ca++-incorporation and spike activity in longitudinal smooth muscle of cat jejunum

Summary Prostaglandin E₁ (0.3 M) decreased both the⁴⁵Ca⁺⁺-incorporation and the spike activity in isolated longitudinal smooth muscle preparations of the cat jejunum probably by an inhibition on the Ca⁺⁺ influx.     

May K+ ions stimulate the formation of cyclic AMP in the brain independently on their depolarizing action?

Summary The effect of potassium ions on the formation of adenosine 3,5-monophosphate (cAMP) in the rat cerebral cortex in vivo was studied under conditions where development of spreading depression had been blocked by pretreatment of the cerebral cortex by topically applied magnesium ions. A linear relationship between potassium concentrations applied to the cortical surface and levels of cAMP has been found. Moreover, potentiation of the K⁺-effect by magnesium ions has been observed.     

Zinc antagonizes the effect of botulinum type A toxin at the mouse neuromuscular junction

Summary Zn²⁺ (10–100 M) elevated the frequency of miniature end-plate potentials (MEPPs) in the mouse diaphragm. The effect did not depend on external Ca²⁺. Botulinum type A toxin (BTX_A, 50 ng/ml) abolished MEPPs almost completely within 30 min. Zn²⁺ (100 M) restored MEPPs and increased their frequency after they had been abolished by BTX_A in Ca²⁺-free solutions. The antagonistic effect of Zn²⁺ in the Ca²⁺-free solution was reduced by exposing the diaphragm to the toxin in the Ca²⁺-free solutions containing high K⁺. Thus, the action of BTX_A is probably enhanced by depolarization of the motor nerve terminals.     

Mechanism of inhibition of IgE-dependent histamine release from rat mast cells by xestobergsterol A from the Okinawan marine spongeXestospongia bergquistia

Histamine release from rat peritoneal mast cells induced by anti-IgE was essentially complete within 4–5 min. Xestobergsterol A and B, which are constituents of the Okinawan marine spongeXestospongia bergquistia Fromont, dose-dependently inhibited anti-IgE-induced histamine release from rat mast cells. The IC₅₀ values of xestobergsterol A and B for histamine release in mast cells activated by anti-IgE were 0.07 and 0.11 M, respectively. Anti-IgE stimulated PI-PLC activity in a mast cell membrane preparation. Xestobergsterol A dose-dependently inhibited the generation of IP3 and membrane-bound PI-PLC activity. Moreover, xestobergsterol A inhibited Ca²⁺-mobilization from intracellular Ca²⁺-stores as well as histamine release in mast cells activated by anti-IgE. On the other hand, xestobergsterol B did not inhibit the membrane-bound and cytosolic PI-PLC activity, IP3 generation or the initial rise in [Ca²⁺]_i in mast cells activated by anti-IgE. These results suggest that the mechanism of inhibition by xestobergsterol A of the initial rise in [Ca²⁺]_i, of the generation of IP3, and of histamine release induced by anti-IgE, was through the inhibition of PI-PLC activity.     

α1-Adrenergic stimulation of ketogenesis and fatty acid oxidation is associated with inhibition of lipogenesis in rat hepatocytes

Summary The effect of norepinephrine on fatty acid synthesis (³H₂O incorporation into fatty acids), on fatty acid oxidation to CO₂ and on ketogenesis was studied in isolated hepatocytes of fed rats. After incubation with norepinephrine (50 M), lipogenesis was lower (5.7±1.1 nmoles³H₂O incorporated into fatty acids/mg dry weight/30 min) than in controls (7.5±1.7; n=6, p<0.02). In contrast, (1-¹⁴C) palmitate conversion into total ketone bodies was increased to 10.9±1.8 nmoles/mg/30 min with norepinephrine, vs 8.5±1.6 in controls (p<0.05), and more (1-¹⁴C) palmitate was converted to¹⁴CO₂ with norepinephrine than in controls (1.48±0.10 nmoles/mg/30 min vs 1.06±0.11, p<0.05). The inhibitory effect of norepinephrine on lipogenesis was abolished by addition of the ₁-receptor blocker prazosin, but not by ₂ or -blockers. The results demonstrate that the ketogenic effect of norepinephrine is coupled with an inhibitory effect on lipogenesis which may be explained by diminished activity of acetyl-CoA carboxylase, diminished formation of malonyl-CoA and decreased activity of carnitine palmitoyl transferase I.     

β-phenylethyl isothiocyanate-mediated apoptosis in hepatoma HepG2 cells

-Phenylethyl isothiocyanate (PEITC) is a promising chemoprotective compound that is routinely consumed in the diet as its glucosinolate precursor. Previous studies have shown that PEITC can inhibit phase I enzymes and induce phase II detoxification enzymes along with apoptosis in vitro. The detailed mechanisms involved in the apoptotic cascade, however, have not been elucidated. In the present study, we demonstrate that PEITC can induce apoptosis in hepatoma HepG2 cells in a concentration- and time-dependant manner as determined by TUNEL positive and SubG1 population analysis. Caspase-3-like activity and poly(ADP-ribosyl)polymerase cleavage increased during treatment with 20 µM PEITC; high concentrations, however, induced necrosis. Pre-treatment with Z-VAD-FMK and the caspase-3-specific inhibitor Ac-DEVD-CHO prevented PEITC-induced apoptosis, as determined by caspase-3-like activity and DNA fragmentation. Additional investigations also showed that at concentrations of 5-C10 µM PEITC, DNA synthesis was inhibited and G2/M phase cell cycle arrest occurred, correlating with an alteration in cyclin B1 and p34^cdc2 protein levels. Furthermore, we also demonstrate a concentration- and time-dependant burst of superoxide (O₂^-) in PEITC-treated cells. However, pre- and co-treatment with the free radical scavengers Trolox, ascorbate, mannitol, uric acid and the superoxide mimetic manganese (III) tetrakis (N-methyl-2-pyridyl) porphyrin failed to prevent PEITC-mediated apoptosis. Taken together, these results suggest that PEITC potently induces apoptosis and cell cycle arrest in HepG2 cells and that the generation of reactive oxygen species appears to be a secondary effect.Received 23 December 2002; accepted 22 April 2003     

Role of calcium in the histamine release induced by D-galactosamine from rat mast cells

Summary Rat peritoneal mast cells were isolated and purified by differential centrifugation in Ficoll. Cells pooled from three to four rats were suspended at approximately 10⁶ cells/ml in a buffered salt solution and incubated for 1 h at 37°C in 300 l volumes in the absence or presence (9×10^–4 M) of calcium chloride. Addition of D-galactosamine hydrochloride (DGM; 2.8×10^–4 M) caused (in addition to basal release) a mean ±SEM percent histamine release of 15.7±5.2 in the presence of Ca⁺⁺ and 19±4.9 in the absence of Ca⁺⁺ (p>0.05). It is suggested that D-galactosamine does not require extracellular Ca⁺⁺ for the release of histamine from the rat mast cell.A preliminary analysis of these results was presented at the International Symposium on calcium entry blockers and tissue protection, Rome, 15–16 March 1984.     

Complex formation betweenγ-immunoglobulin and calmodulin in calcium-free conditions

We show that -immunoglobulin (IgG) binds calmodulin (CaM) in a Ca²⁺-independent manner, with Kd value of (1.7±0.5)×10^–7M. A single IgG molecule maximally bound 10 CaM molecules. The binding is to the heavy chain or Fab portion, but not the Fc portion, of the IgG molecules. Ca²⁺ greatly diminished the interaction between IgG and CaM, with IC₅₀=8–9M. These data give a novel insight into protein-protein interactions.     

Effect of clofibrate on the enzyme activity of rat liver plasma membranes

Summary The activity of 3 plasma membranes marker enzymes (5-nucleotidase, Mg⁺⁺-ATPase and alkaline phosphodiesterase-I) was determined in plasma membranes isolated from liver of control and of clofibrate-treated rats. A complete identity of plasma membranes enzyme activity in the 2 groups of experimental animals was observed for the 3 enzymes studied.     

Self-labeling of human polymorphonuclear leucocyte myeloperoxidase with125iodine

In order to obtain a radioimmunoassay (RIA) technique for the measurement of human plasma myeloperoxidase (MPO), we purified the enzyme from polymorphonuclear granulocytes (neutrophils), and compared three methods of labeling it with¹²⁵Iodine: chloramine T, lactoperoxidase, and an original technique of self labeling based on the ability of the enzyme to oxidize and bind¹²⁵I in the presence of H₂O₂. The chloramine T technique produced a degraded protein, as well shown by a high non-specific binding of tracer to antibody. The lactoperoxidase technique did not succeed in labeling MPO with an adequate specific activity. In contrast, the self-labeling method gave a stable tracer with a specific activity of 23 Ci/gmg MPO (85 MBq), a satisfactory level of immunoreactivity, and a low-specific binding (3%). After labeling, purification of tracer was achieved by gel filtration chromatography in phosphate buffer (0.05 M; pH7) to which 0.1% poly-L-lysine was added. The labeled molecule remained stable for 40 days and could be used for RIA with a polyclonal antibody raised in rabbits.     

Calcium release from frog sarcoplasmic reticulum by an imidazolyl reagent

Summary Calcium is released from the isolated heavy sarcoplasmic reticulum (SR) of frog skeletal muscle upon application of 0.1–1 mM diethylpyrocarbonate (DEP, an imidazolyl reagent). The Ca-ATPase activity of SR was suppressed by 20% in the presence of 1 mM DEP. More than 1 mM of free magnesium ion or 5 M ruthenium red eliminated the effect of DEP on calcium release but not on Ca-ATPase activity. A plausible site of DEP action is on the calcium channel.     

C-peptide stimulates Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase via activation of ERK1/2 MAP kinases in human renal tubular cells

Proinsulin-connecting peptide (C-peptide) exerts physiological effects partially via stimulation of Na⁺, K⁺-ATPase. We determined the molecular mechanism by which C-peptide stimulates Na⁺, K⁺-ATPase in primary human renal tubular cells (HRTCs). Incubation of the cells with 5 nM human C-peptide at 37°C for 10 min stimulated ⁸⁶Rb⁺ uptake by 40% (p<0.01). The carboxy-terminal pentapeptide was found to elicit 57% of the activity of the intact molecule. In parallel with ouabain-sensitive ⁸⁶Rb⁺ uptake, C-peptide increased subunit phosphorylation and basolateral membrane (BLM) abundance of the Na⁺, K⁺-ATPase ₁ and ₁ subunits. The increase in BLM abundance of the Na⁺, K⁺-ATPase ₁ and ₁ subunits was accompanied by depletion of ₁ and ₁ subunits from the endosomal compartments. C-peptide action on Na⁺, K⁺-ATPase was ERK1/2-dependent in HRTCs. C-peptide-stimulated Na⁺, K⁺-ATPase activation, phosphorylation of ₁-subunit and translocation of ₁ and ₁ subunits to the BLM were abolished by a MEK1/2 inhibitor (20 M PD98059). C-peptide stimulation of ⁸⁶Rb⁺ uptake was also abolished by preincubation of HRTCs with an inhibitor of PKC (1 M GF109203X). C-peptide stimulated phosphorylation of human Na⁺, K⁺-ATPase subunit on Thr-Pro amino acid motifs, which form specific ERK substrates. In conclusion, C-peptide stimulates sodium pump activity via ERK1/2-induced phosphorylation of Thr residues on the subunit of Na⁺, K⁺-ATPase.Received 15 June 2004; received after revision 14 September 2004; accepted 14 September 2004     

Inhibitory effect of fasting on the glucagon-induced increase of liver phosphorylase A activity in rats

Summary The increase of liver phosphorylase A activity observed 1, 2 and 3 min after i.v. administration of 0.1 g kg^–1 glucagon to fed rats was found to be completely absent in 24 h fasted animals, although there is even an exaggerated liver cAMP response to glucagon after fasting.Acknowledgments. We would like to thank Mrs M. Fritzová and Mr F. Kovai for expert technical assistance.