Volatile anesthetics inhibit NMDA-stimulated45Ca uptake by rat brain microvesicles

We have previously shown that volatile anesthetics inhibit glutamate-stimulated [³H]MK-801 binding to the ionophore of NMDA receptor complexes in rat brain. In the present study, we examined the influence of enflurane and halothane on NMDA-stimulated⁴⁵Ca uptake by a microvesicle fraction isolated from rat brain. NMDA stimulated⁴⁵Ca uptake (30 sec) by rat brain microvesicles by up to 70% with an EC₅₀ of 1.4±0.5 M. The NMDA-stimulated⁴⁵Ca uptake was inhibited by MK-801 and D-AP-5 with IC₅₀'s of 10 M. Enflurane and halothane inhibited⁴⁵Ca uptake stimulated by 100 M NMDA by as much as 60–80% with IC₅₀'s of 0.2–0.3 mM, concentrations achieved during routine clinical use. Basal⁴⁵Ca uptake measured in the absence of agonist was not affected by the anesthetics. Glycine did not affect the level of NMDA-stimulated⁴⁵Ca uptake, but markedly reduced the inhibition of uptake caused by enflurane and halothane. Preincubation of microvesicles with NMDA resulted in a desensitization of NMDA-stimulated⁴⁵Ca uptake, with a t_1/2 of 20 sec. Enflurane and halothane diminished both the extent and rate of development of this desensitization, as did glycine. These findings support the idea that volatile anesthetic interference with neurotransmission at NMDA receptor complexes contributes to the development of the anesthetic state.     

Pancreastatin activates pertussis toxin-sensitive guanylate cyclase and pertussis toxin-insensitive phospholipase C in rat liver membranes

We have recently found the calcium dependent glycogenolytic effect of pancreastatin on rat hepatocytes and the mobilization of intracellular calcium. To further investigate the mechanism of action of pancreastatin on liver we have studied its effect on guanylate cyclase, adenylate cyclase, and phospholipase C, and we have explored the possible involvement of GTP binding proteins by measuring GTPase activity as well as the effect of pertussis toxin treatment of plasma liver membranes on the pancreastatin stimulated GTPase activity and the production of cyclic GMP and myo-inositol 1,4,5-triphosphate. Pancreastatin stimulated GTPase activity of rat liver membranes about 25% over basal. The concentration dependency curve showed that maximal stimulation was achieved at 10^?7 M pancreastatin (EC₅₀ = 3 nM). This stimulation was partially inhibited by treatment of the membranes with pertussis toxin. The effect of pancreastatin on guanylate cyclase and phospholipase C were examined by measuring the production of cyclic GMP and myo-inositol 1,4,5-triphosphate respectively. Pancreastatin increased the basal activity of guanylate cyclase to a maximum of 2.5-fold the unstimulated activity at 30°C, in a time- and dose-dependent manner, reaching the maximal stimulation above control with 10^?7 M pancreastatin at 10 min (EC₅₀ = 0.6 nM). This effect was completely abolished when rat liver membranes had been ADP-ribosylated with pertussis toxin. On the other hand, adenylate cyclase activity was not affected by pancreastatin. Phospholipase C activity of rat liver membranes was rapidly stimulated (within 2–5 min) at 30°C by 10^?7 M pancreastatin, reaching a maximum at 15 min. The dose response curve showed that with 10^?7 M pancreastatin, maximal stimulation was obtained (EC₅₀ = 3 nM). GTP (10^?5 M) stimulated the membrane-bound phospholipase C as expected. However, the incubation of rat liver membranes with GTP partially inhibited the stimulation of phospholipase C activity produced by pancreastatin, whereas GTP enhanced the activation of phospholipase C by vasopressin. This inhibition by GTP was dose dependent and 10^?5 M GTP obtained the maximal inhibition (about 40%). the inhibitory effect of GTP on the stimulatory effect of pancreastatin on phospholipase C activity was completely abolished when rat liver membranes had previously been ADP-ribosylated with pertussis toxin. The presence of 8-Br-cGMP mimics the effect of GTP, whereas GMP-PNP increased both basal and pancreastatin-stimulated phospholipase C, suggesting a role of the cyclic GMP as a feed-back regulator of the synthesis of myo-inositol 1,4,5-triphosphate. However, the pretreatment of membranes with pertussis toxin did not modify the production of myo-Inositol 1,4,5-triphosphate stimulated by pancreastatin. In conclusion, pancreastatin activates guanylate cyclase activity and phospholipase C involving different pathways, pertussis toxin-sensitive, and -insensitive, respectively. © 1994 Wiley-Liss, Inc.     

A nonradioactive method for isolating complementary DNAs endocing calmodulin-binding proteins

Calmodulin labeled with¹²⁵I or³⁴S has been used to screen expression libraries to isolate cDNAs encoding calmodulin-binding proteins (CBPs) from several eukaryotic systems. The use of radiolabeled calmodulin has, however, several disadvantages. We have developed a nonradiactive method to isolate cDNAs for CBPs using biotinylated calmodulin. Screening of a cDNA library in an expression vector with biotinylated calmodulin resulted in the isolation of cDNAs encoding CBPs. Avidin and biotin blocking steps, prior to incubation of the filters with biotinylated calmodulin, are found to be essential to eliminate the cDNAs that code for biotin-containing polypeptides. The cDNA clones isolated using this nonradioactive method bound calmodulin in a calcium-dependent manner. The binding of biotinylated calmodulin to these clones was completely abolished by ethylene glycolbis(\-aminoethylether)-N,N′-tetraacetic acid (EGTA), a calcium chelator. Furthermore, the isolated cDNAs were confirmed by probing the clones with³⁵S-labeled calmodulin. All the isolated clones bound to radiolabeled calmodulin in the presence of calcium but not in the presence of EGTA. The method described here is simple, fast, and does not involve preparation and handing of radiolabeled calmodulin. All the materials used in this method are commercially available; hence, this procedure should be widely applicable to isolate cDNAs encoding CBPs from any eukaryotic organism.     

Modulation of fibroblast response to maitotoxin along the cell division cycle

Maitotoxin (MTX) induces an increase of [Ca²⁺]_i and of phosphoinositide breakdown in various cell types. The [Ca²⁺]_i increase followed with fluorescent probes on cell suspensions has been described as slow and lasting, in contrast to the signal induced by calcium ionophores such as ionomycin. MTX effects have been studied on two fibroblastic cell lines, BHK21 C13 and FR 3T3, synchronized by serum deprivation treatment performed in an isoleucine-free medium for BHK21 C13 cells. In BHK21 C13 cells, flow cytometry analysis showed that two stages, G1/S and G2/M, were particularly susceptible to MTX treatment. Scanning laser cytometry demonstrated that calcium response of FR 3T3 fibroblasts followed with Indo-1 varied during the cell division cycle. The [Ca²⁺]_i increase was almost always vertical, but its delay after MTX addition lasted from zero (S and G2/M transition) to 10–20 min (G1) or more (G2). No [Ca²⁺]_i change could be detected during mitosis. The [Ca²⁺]_i response at the S phase was biphasic. These observations suggest that (1) the lasting response described in the literature represents a global cell population effect, and (2) cells are more sensitive to MTX at specific stages of the cell division cycle, which could correspond to periods when calcium signals have been detected in different cell types.Abbreviations MTX maitotoxin - [Ca²⁺]_i intracellular calcium concentration - IP₃ inositol triphosphate     

Activation of murine macrophages by hydrogen peroxide

Hydrogen peroxide at concentrations from 0.1 to 20 μM enhances phagocytosis and oxidative burst of murine peritoneal macrophages. The activation of these macrophage functions is paralled by prolonged hyperpolarization and a transient increase in cytoplasmic free calcium concentration. All the effects are dose- and time-dependent. The results obtained for H₂O₂ are compared with those for a natural activator, peptide N-formyl-methionyl-leucly-phenylalanine. The data demonstrate the ability of small doses of hydrogen peroxide to stimulate macrophages through the intracellular mechanisms of ion transduction.     

Influence of endotoxin on the stereoselective pharmacokinetics of oxprenolol,propranolol, and verapamil in the rat

The influence of endotoxin-induced inflammation on the enantioselective pharmacokinetics of propranolol, oxprenolol, and verapamil, which bind to α₁-acid glycoprotein, was studied in the rat. The racemic mixtures were given orally. In the control animals, for propranolol and oxprenolol, the plasma concentrations of the (R)-enantiomer were higher than those of the (S)-enantiomer, while for verapamil the reverse was true. Protein binding and intrinsic clearance are the main factors responsible for this enantioselectivity. After endotoxin treatment, for the three drugs tested the plasma concentrations and the plasma binding of both enantiomers were significantly increased. This effect was more pronounced for (R)-propranolol, (R)-oxprenolol, and (S)-verapamil than for their respective antipodes. The enantioselective effect of endotoxin on the plasma concentrations of the drugs studied seems mainly due to the enantioselective increase in binding to α₁-acid glycoprotein. © 1994 Wiley-Liss, Inc.     

Accumulation of calcium, potassium and magnesium in apple fruits under various conditions of humidity

In each of two experiments done under controlled conditions, starting at bloom, 4 humidity treatments were applied to potted trees of apple ( Malus pumila Mill. cv. Cox's Orange Pippin), i. e. in experiment 1: (1) high humidity throughout, (2) low humidity throughout, (3) low humidity for 7 weeks followed by high humidity for 6-7 weeks, and (4) the reverse (first high and then low humidity); in experiment 2: (1) day/night humidity high/high, (2) low/low, (3) low/high, and (4) high/low.
In both experiments high humidity favoured shoot growth appreciably. Change from low to high humidity after 7 weeks resulted in some growth stimulation but in the reverse situation growth was markedly reduced. Shoot growth responded little to different night humidities. In the two experiments fruit growth was little affected by treatments. In experiment 2 irrespective of night humidity, water consumption was higher at low than at high day humidity. In the high/low humidity regime water use during the night was high and leaf water potential low, relatively; during the day water potential was little affected by treatments.
At any time leaf Ca and Mg were clearly highest at low day humidity; night humidity had no effect. Leaf K did not respond to treatments. Fruit Ca at high humidity throughout was lower than at low humidity throughout. Increasing humidity later in the season was ineffective but a decrease at that time tended to reduce fruit Ca slightly. The clearly lowest Ca values occurred in the high/low day/night treatment. Fruit K and Mg were not or hardly affected by treatments.
The findings are discussed in terms of humidity effects on transpiration, shoot and fruit growth, and xylem mineral concentration and ion exchange translocation along the xylem walls.     

Interactions between aluminium,magnesium and calcium with different monocotyledonous and dicotyledonous plant species

Growth inhibition of plants suffering from Al toxicity is generally accompanied by impaired root development which can be quantitatively described by reduced specific root length (m g^-1 dry root). In addition, the uptake of nutrients such as Mg and Ca is inhibited. Increased supply of either Mg or Ca can significantly diminish the negative effect of Al on root development and improve the Mg or Ca nutrition of the plants. The positive effect of Ca is well established but the effect of Mg has been observed in only a few plan species. Therefore, the effects of increasing Mg and Ca supply on Al toxicity in plants of seven monocots and eight dicots have been now examined in nutrient solution experiments. In general, Mg appears to be more effective than Ca in alleviating Al toxicity with the monocots, whereas the reverse is true for the dicots. Increased concentrations of Mg and Ca in solution seem to protect the plants against Al toxicity by improving the Mg or Ca nutrition and by alleviating the toxic effect of Al on root development.     

Improving the mineral reserves and protein quality of maize (Zea mays L.) kernels using unique genes

The effects of the maize genes, o ₂ and Mal, on the concentrations of mineral nutrient cations and amino acid levels in mature maize (Zea mays L) kernels of various inbred lines were studied. Previously, the o ₂ gene has been used to improve the protein quality and increase the mineral nutrient content of kernels from some inbred lines. Genotypes possessing the Mal (multiple aleurone layer) gene, contain more than one row of aleurone cells in their kernels and this gene enhances the effect of the o ₂gene on improving kernel protein quality. Incorporating these genes into the maize genome increased accumulation of several mineral nutrients (including Ca, Mg, Zn, Fe, Mn, Zn and Cu) in some of the experimental lines studied. The physiological basis for this increase of mineral nutrients in the kernels is discussed. The effect of the Mal gene on the kernel amino acid composition and protein quality was also examined. Possibly, these genes could be used in combination in breeding programs to improve kernel quality and nutritional value of maize.     

NAD(P)H-ubiquinone oxidoreductases in plant mitochondria

Plant (and fungal) mitochondria contain multiple NAD(P)H dehydrogenases in the inner membrane all of which are connected to the respiratory chain via ubiquinone. On the outer surface, facing the intermembrane space and the cytoplasm, NADH and NADPH are oxidized by what is probably a single low-molecular-weight, nonproton-pumping, unspecific rotenone-insensitive NAD(P)H dehydrogenase. Exogenous NADH oxidation is completely dependent on the presence of free Ca²⁺ with aK _0.5 of about 1 µM. On the inner surface facing the matrix there are two dehydrogenases: (1) the proton-pumping rotenone-sensitive multisubunit Complex I with properties similar to those of Complex I in mammalian and fungal mitochondria. (2) a rotenone-insensitive NAD(P)H dehydrogenase with equal activity with NADH and NADPH and no proton-pumping activity. The NADPH-oxidizing activity of this enzyme is completely dependent on Ca²⁺ with aK _0.5 of 3 µM. The enzyme consists of a single subunit of 26 kDa and has a native size of 76 kDa, which means that it may form a trimer.