Studies on the effects of insulin and acetylcholine on activation of glycogen synthase and on glycogenesis in hepatocytes

The addition of insulin (4.0 × 10^?11 M) or acetylcholine (10^?6 M) to isolated hepatocytes stimulated glycogen accumulation and this stimulation was more pronounced when the medium glucose was raised from 50 to 300 mg percent. Studies with [¹⁴C]-glucose showed a two-fold stimulation in glycogen synthesis by the addition of insulin (4.0 × 10^?11 M) or acetylcholine (10^?6 M). A sixteen percent increase in the activity of glycogen synthase was observed in cells incubated for 10 minutes with insulin (4.0 × 10^?11 M) or acetylcholine (10^?6 M), whereas at one hour incubation a 40 percent increase in activity was observed with the same concentration of insulin or acetylcholine. The effects of insulin and acetylcholine were not additive.     

Anticonvulsants and trifluoperazine inhibit the evoked release of GABA from cerebral cortex of rat at different sites

The action of anticonvulsant drugs, phenytoin, diazepam, clonazepam and phenobarbitone, was tested on the release of [¹⁴C]-GABA from tissue slices of rat cerebral cortex. All drugs caused a significant dose-dependent depression of the 33mM-K⁺-evoked release of [¹⁴C]-GABA but had little effect on the resting release of [¹⁴C]-GABA, except at high concentrations. The IC₅₀ values for inhibition of K⁺-evoked release of [¹⁴C]-GABA were 4.7 × 10^?5, 7 × 10^?5, 28 × 10^?5 and 7.9 × 10^?4M for diazepam, clonazepam, phenytoin and phenobarbitone respectively. Trifluoperazine also caused a similar and complete inhibition of [¹⁴C]-GABA release with an IC₅₀ of 1 × 10^?5M. The effect of diazepam and trifluoperazine were additive. The inhibition by trifluoperazine could be overcome by addition of exogenous calmodulin, whereas that of diazepam, phenytoin or phenobarbitone was not overcome. It is proposed that the anticonvulsants tested inhibit calcium-dependent transmitter release at a site distal to the formation of a calcium-calmodulin complex, which is presumably activated by this complex. Trifluoperazine, on the other hand, acts by reducing the availability of calmodulin.     

Effect of 2-deoxy-d-Glucose on Aminoacids Metabolism in Rats’ Cerebral Cortex Slices

We studied the effect of different concentrations of 2-deoxy-d-glucose on the l-[U-¹⁴C]leucine, l-[1-¹⁴C]leucine and [1-¹⁴C]glycine metabolism in slices of cerebral cortex of 10-day-old rats. 2-deoxy-d-glucose since 0.5 mM concentration has inhibited significantly the protein synthesis from l-[U-¹⁴C]leucine and from [1-¹⁴C]glycine in relation to the medium containing only Krebs Ringer bicarbonate. Potassium 8.0 mM in incubation medium did not stimulate the protein synthesis compared to the medium containing 2.7 mM, and at 50 mM diminishes more than 2.5 times the protein synthesis compared to the other concentration. Only at the concentration of 5.0 mM, 2-deoxy-d-glucose inhibited the CO₂ production and lipid synthesis from l-[U-¹⁴C] leucine. This compound did not inhibit either CO₂ production, or lipid synthesis from [1-¹⁴C]glycine. Lactate at 10 mM and glucose 5.0 mM did not revert the inhibitory effect of 2-deoxy-d-glucose on the protein synthesis from l-[U-¹⁴C]leucine. 2-deoxy-d-glucose at 2.0 mM did not show any effect either on CO₂ production, or on lipid synthesis from l-[U-¹⁴C]lactate 10 mM and glucose 5.0 mM.     

The fate of [14C]glucose during cold-hardening in Eurosta solidaginis (Fitch)

Glucose catabolism in overwintering larvae Eurosta solidaginis was examined to determine the relative contributions of glycolysis and the pentose phosphate pathway to polyol synthesis at different temperatures. Rates of ¹⁴CO₂ evolution were determined after injection of [¹⁴C]1-glucose, [¹⁴C]6-glucose, and [¹⁴C]3,4-glucose. In addition incorporation of label from each isotope into sorbitol and glycerol was monitored. The respirometric studies showed a relative increase in pentose phosphate activity between 10 and 5°C. Similar results were obtained from the changes of radioactivities incorporated into glycerol, although the activation of the pentose phosphate pathway was low. The conversion of [¹⁴C]glucose to glycerol was highest at 10°C, suggesting that maximum glycerol synthesis may occur at this temperature. Radioactivity appeared in the sorbitol fraction of larvae incubated at temperatures below 5°C. Late autumn larvae converted more [¹⁴C]glucose than did early autumn larvae.     

Presynaptic Control of the Synthesis and Release of Dopamine from Striatal Synaptosomes: A Comparison Between the Effects of 5-Hydroxytryptamine, Acetylcholine, and Glutamate

The effects of 5-HT and glutamate on dopamine synthesis and release by striatal synaptosomes were investigated and compared with the action of acetylcholine, which acts presynaptically on this system. 5-HT inhibited (28%) synthesis of [¹⁴C]dopamine from L-[U-¹⁴C]tyrosine, at 10-⁵M and above. This contrasts with the action of acetylcholine, which stimulated [¹⁴C]-dopamine synthesis by 24% at 10-⁴ M. Tissue levels of GABA were unaffected by either 5-HT or acetylcholine up to concentrations of 10-⁴ M. The inhibitory action of 5-HT (5 × 10^?5 M and 2 × 10^?4 M) on [¹⁹C]dopamine synthesis was completely abolished by methysergide (2 × 10^?6 M). Higher concentrations of methysergide (10^?4 M) or cyproheptadine (10^?5 M) inhibited [¹⁴C]dopamine synthesis by 28% and 25%, respectively, when added alone to synaptosomes. However, only methysergide prevented the further inhibition of synthesis caused by 5-HT. At concentrations of 2 × 10^?5 M and above, 5-HT stimulated [¹⁴C]dopamine release. This releasing action differed from that of acetylcholine, which occurred at lower concentrations (e.g., 10^?6 M). Methysergide (up to 10^?4 M) or cyproheptadine (2 × 10^?4 M) did not reduce the 5-HT (5 × 10^?5 M)-induced release of [¹⁴C]dopamine, but methysergide (10^?4 M) showed a potentiation (49%) of this increased release. The stimulatory effects of 5-HT (2 × 10^?5 M) and K⁺ (56 mM) on [¹⁴C]dopamine release were additive, indicating that two separate mechanisms were involved. However, when both agents were present the stimulatory effect of K⁺ (56 mM) on [¹⁴C]dopamine synthesis was not seen above the inhibitory effect of 5-HT. Glutamate (0.1-5 mM) did not affect [⁴C]dopamine release or its synthesis from L-[U-¹⁴C]tyrosine. It is concluded that 5-HT modulates the synthesis of dopamine in striatal nerve terminals through a presynaptic receptor mechanism, an action antagonised by methysergide. The releasing action of 5-HT apparently occurs through a separate mechanism which is also distinct from that involved in the response to K⁺ depolarisation.     

A simple method for determining total glucose utilisation by isolated adipocytes using (5-3H)-glucose

Glucose utilisation by adipocytes incubated with and without insulin and at two concentrations of extracellular glucose has been estimated by three different procedures. Glucose disappearance from the medium was calculated by using glucose oxidase to determine the glucose concentration remaining after incubation and comparing this with the glucose concentration in standard solutions made up by appropriate dilution of the original medium. [U-¹⁴C]-glucose utilisation was calculated by summing the ¹⁴C found in CO₂, triglycerides, and anions. [³H]-H₂O formation from [5-³H]-glucose was the third measure of glucose utilisation. All three methods gave similar answers, but the [5-³H]-glucose is simpler to use than [U-¹⁴C]-glucose and gives substantially more reproducible results than glucose oxidase.     

SR 140333, a Novel, Selective, and Potent Nonpeptide Antagonist of the NK1 Tachykinin Receptor: Characterization on the U373MG Cell Line

The effects of a novel nonpeptide NK1 tachy-kinin receptor antagonist, SR 140333, on the functional consequences of NK1 receptor activation in a human astrocytoma cell line, U373MG, were investigated. Radioligand binding conducted with ¹²⁵l-Bolton-Hunter substance P revealed a competitive inhibition by SR 140333 and its R enantiomer SR 140603 with K_i values of 0.74 and 7.40 nM, respectively. The NK1-selective agonist, [Sar⁹,Met(O₂)¹¹]-substance P, stimulated the formation of inositol phosphates with an EC₅₀ of 3.8 × 10^?9M. SR 140333 blocked the stimulatory effect of this agonist (10^?7M) with an IC₅₀ of 1.6 × 10^?9M,whereas the effect of another NK1 agonist, septide (EC₅₀= 1.5 × 10^?8M)was antagonized with an IC₅₀ of 2.1 × 10^?10M.Enhancement of [³H]taurine release by [Sar⁹,Met(O₂)¹¹]-substance P (EC₅₀= 7.4 × 10^?9M) was also inhibited by SR 140333 with an IC₅₀ of 1.8 × 10^?9 M. SR 140603 was 10-fold less potent than SR 140333 in inhibiting inositol monophosphate formation and [³H]taurine release. The calcium mobilization induced by [Sar⁹,Met(O₂)¹¹]-substance P (10^?8M) was totally prevented by 10^?8MSR 140333. Patchclamp experiments showed that SR 140333 depressed the outward current evoked by 5 × 10^?8M [Sar⁹, Met(O₂)¹¹]-substance P with an IC₅₀ of 1.3 × 10^?9M. The expression of c-fos was stimulated by [Sar⁹,Met(O₂)¹¹]-substance P with an EC₅₀ of 2.5 × 10^?10M, an effect that was also inhibited by SR 140333 with an IC₅₀ of 1.1 × 10^?9M. The present results illustrate the sequential events of the response elicited by NK1 agonists, which were antagonized by SR 140333, demonstrating its powerful NK1 antagonist activity on a functional basis.     

Characterization of prostaglandin F2α receptor of mouse 3T3 fibroblasts and its functional expression in Xenopus laevis oocytes

Prostaglandin (PG) F_2α increased [³H]thymidine incorporation into quiescent NIH 3T3 cells, stimulated phosphoinositide breakdown, and raised intracellular Ca²⁺ concentration ([Ca²⁺]_i) in a dose-dependent manner with ED₅₀ values of 2.0 × 10^?8 M, 4.6 × 10^?8 M, and 7.5 × 10^?8 M, respectively. The increase in [³H]thymidine incorporation with PGF_2α was additive with that seen with epidermal growth factor (EGF) or insulin. The peak [Ca²⁺]_i increase with PGF_2α was still obvious in the absence of extracellular Ca²⁺ and was insensitive to islet activating protein (IAP) pretreatment. Membranes prepared from NIH 3T3 cells exhibited a specific binding for PGF_2α, which was sensitive to GTP_γS but not sensitive to IAP pretreatment. Xenopus laevis oocytes injected with NIH 3T3 cell mRNA between 18S and 28S rRNA fractionated by sucrose gradient, expressed a PGF_2α-specific Cl^? current when examined by voltage clamp. This Cl^? current was also insensitive to IAP pretreatment and not affected by extracellular Ca²⁺ concentration ([Ca²⁺]_o). These results indicate 1) that the NIH 3T3 cells expressed a specific PGF_2α receptor which is linked to phosphoinositide-specific phospholipase C (PLC) activation and to mobilization of Ca²⁺ via an IAP-insensitive G-proteins(s), 2) that this PGF_2α receptor may play an active role in the proliferation of NIH 3T3 cells, and 3) that this PGF_2α receptor can be expressed in the oocyte system. © 1993 Wiley-Liss, Inc.     

Biphasic toxicity of diethyldithiocarbamate, a metal chelation, to T lymphocytes and polymorphonuclear granulocytes: reversal by zinc and copper.

A new type of toxicity biphasically dependent on concentration was observed with diethyldithiocarbamate, a metal chelator utilized in medicine. As judged by cell survival and [³H]Urd incorporation, diethyldithiocarbamate was maximally toxic to T lymphocytes and polymorphonuclears at 2.5×10^?5 M (first phase) and at higher than 2.5×10^?3 M (second phase), but was not toxic at intermediate concentrations around 2.5×10^?4 M. The response of chelator treated T lymphocytes to phytohemagglutinin was also biphasic. The first toxic phase was partially reversed by 2.5×10^?5 M ZnCl₂, while the second phase was partially reversed by 10^?2 M CuCl₂. This suggests that inhibition of Zn-metalloenzymes in the first phase and of Cu-metalloenzymes in the second may play a crucial role in the mechanism of toxicity. The second toxic phase may be in part due to the observed inhibition of superoxide dismutase rendering the cells susceptible to oxygen toxicity, like obligate anaerobes.     

Regulation of morphological and functional properties of astrocytes by hydrogen peroxide

Effects of hydrogen peroxide on morphological characteristics, proliferation index, and menadione-dependent lucigenin-enhanced chemiluminescence of C6 glioma cells were studied. It was established that H₂O₂ at 5 × 10^?7?1 × 10^?8 M concentrations acted as a regulator of morphological and functional properties of astrocytes, inducing their reactivation, which is manifested as cell body hypertrophy and an increase of proliferative activity and menadione-induced production of superoxide anion radicals (O ₂ ^?? ). Cytodestructive action of hydrogen peroxide at a concentration higher than 1 × 10^?6 M on C6 glioma cells shows itself as a decrease of their proliferation index and the ability to generate O ₂ ^?? under the effect of menadione. Use of lipopolysaccharide B as a functional stimulator has shown that H₂O₂ modifies signaling pathways leading to an increase of mitotic activity of C6 glioma cells and decreases the yield of lucigenin-dependent chemiluminescence of astrocytes under the action of menadione to the level of control values.     

Stimulation of pyruvate dehydrogenase activity in adipocytes by oxytocin: Evidence for mediation of the insulin-like effect by endogenous hydrogen peroxide independent of glucose transport

Oxytocin, a nonapeptide posterior pituitary hormone, which is known to increase glucose oxidation in fat cells like insulin, is shown here to stimulate pyruvate dehydrogenase activity in these cells. The process appears to involve the activation of preexisting molecules since there was no change in the total enzyme content after full activation. The effect of oxytocin, as well as of insulin, appears to be mediated by endogenous H₂O₂ formation, as evident from (i) the enhanced [¹⁴C]formate oxidation and its greater inhibition by 3-amino-1,2,4-triazole in the hormone-treated cells than in the control. This is a measure of the catalase:H₂O₂ complex, and the dose dependence of this response is found to be identical with that of glucose oxidation via the hexose monophosphate shunt pathway and of pyruvate dehydrogenase activity; and (ii) treatment of the cells with low concentration of exogenous H₂O₂ causes the activation of pyruvate dehydrogenase to the extent which is comparable with the effect of the hormones. The ED₅₀ of oxytocin was 7 × 10^?9m, whereas the ED₅₀ of insulin was 5 × 10^?11m. The reduced, inactive (SH) derivatives of the hormones had the same dose-response relationship, but considerably lower effect (10 to 20% of the native molecules of the hormones), indicating the significant role of the disulfide bridge(s) in eliciting these metabolic responses. The stimulation of PDH by oxytocin or insulin is found to be essentially independent of medium glucose which, however, can sustain the response apparently by recycling the intracellular oxidation-reduction state. However, unlike insulin, oxytocin fails to stimulate the rapid uptake of 3-O-[³H]methyl-d-glucose in these cells. The data illustrate that the major metabolic actions of insulin, viz., glucose utilization and lipogenesis, are shared by another heterologous polypeptide hormone, e.g., oxytocin, through a common effector, H₂O₂. It is suggested that (i) oxytocin may play a limited surrogate role for insulin in these cells; and (ii) H₂O₂ production may be the general basis of oxytocin's action.     

Measurement of rate constants for quenching singlet oxygen with a Cypridina luciferin analog (2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazo[1,2-a]pyrazin-3-one) and sodium azide

The rate constants for [¹O₂] [MCLA] and [¹O₂][NaN₃] were measured by quenching the near-infrared emission (¹Δ_g→³∑_g) in steady state with MCLA and NaN₃, respectively. ¹O₂ was constantly generated by energy transfer to O₂ from Ar laser-excited Rose Bengal. The Stern—Volmer plots yielded the second-order rate constants of 2.94 × 10⁹ M^?1 S^?1 and 3.83 × 10⁸ M^?1 S^?1 for quenching ¹O₂ with MCLA and NaN₃ in water at pH 5.4, respectively. The ¹O₂ + MCLA reaction emitted light with maximum at 465 nm at pD 4.5 identical to the O₂^? + MCLA reaction.     

Hydrocortisone binding receptor in the rat pituitary GH3 cell line.

A receptor with specificity and high affinity for hydrocortisone (HC) has been found in the cytosol of GH₃ cells, a growth hormone (GH) producing culture. Scatchard analysis indicated that the interaction of [³H]HC with the receptor has an apparent dissociation constant (K_d) of about 6.0 × 10^?9M and a concentration of binding sites of approx. 1 × 10^?13 mol/mg cytosol protein. The second order association rate constant was determined to be 1.5 × 10⁶ M^?1 min^?1. The receptor activity is stable at 2°C for several hours, but is destroyed completely by heating at 37°C for 1 hour, or by treatment with pronase, only partially by RNase, but not by DNase. The binding of [³H]HC to the cytosol receptor is inhibited by unlabeled progesterone (PR) or dexamethasone to the same extent as the inhibition by unlabeled HC. However, it is only partially inhibited by testosterone, 17-methyl-testosterone, 17α and 17β-estradiol, and 4-pregnen-20β-ol-3-one, and is unaffected by 5α-pregnan-3β,20β-diol. The biological role for these receptors in the regulation of GH synthesis is supported by the observations that the HC-stimulated production of GH is antagonized by PR, which competes with the binding of HC to the receptor.     

17-Hydroxyprogesterone metabolism in the monkey fetal adrenal: C17–20Lyase and 21-hydroxylase activities

C^17–20Lyase and 21-hydroxylase activities were measured during late gestation In the rhesus monkey ($\text{Macaca mulatta}$) fetal adrenal. Activities were assessed in 10,000 × g supernatants with 17-hydroxyprogesterone and NADPH as substrates. Although conversion of [¹⁴C]17-hydroxyprogesterone to [¹⁴C]androstenedione was noted, activity was often nonlinear and far less than the rate of hydroxylation which together prevented an accurate estimation of lyase rate, K_m and V_max. 21-Hydroxylase activity was characterized; the mean reaction rate was 1.6 × 10^?3 μmoles NADPH oxidized/min. × mg^?1 protein with an apparent K_m of 3.6 × 10^?7 M and a V_max of 2.2 × 10^?3 μmoles/min. × mg^?1 protein. These values were similar to data obtained In adrenals from adult monkeys. A relatively high level of hydroxylase activity in the fetal gland might lead to an Inadequate supply of precursors for the synthesis of dehydroepiandrosterone sulfate (DHEAS) in the adrenal if it also contained 3β-hydroxysteroid dehydrogenase (3β-hsdh). However, the fact that the fetal adrenal reportedly is deficient in 3β-hsdh may serve to protect both DHEAS and corticoid synthesis.     

Properties of auricularia auricula-judae β-D-glucan in dilute solution

A Water-soluble glucan A was isolated from the fruit body of Auricularia auricula-judae. It is composed of a backbone chain of β-(1 → 3)-linked D -glucose residues, two out of three glucose residues being substituted at C-6 positions with a single glucose unit. The weight average molecular weight Mw, number average molecular weight Mn, and intrinsic viscosity [η] of the fractionated samples were studied at 25°C in water and in dimethylsulfoxide (DMSO). The Mark-Houwink equation was established as [η] = 6. 10 × 10^?4 Mw^1.14 for the glucan A having Mw ranging from 9 × 10⁵ to 1.6 × 10⁶ in water. The values of [η] in water are far higher than those in DMSO, but the values of Mn measured in water are the same as those in DMSO. Analysis of Mw and [η] in terms of the known theories for rods and wormlike chains yielded 1030 ± 100 nm^?1, 90 ± 20 nm, 1.3 ± 0.3 nm, and 0.26 ± 0.03 nm for molar mass per unit contour length M_L, persistence length q, diameter d, and contour length h per main-chain glucose residue, respectively. The present data suggest that glucan A dissolves in water as single-stranded helical chains and in DMSO as semiflexible chains. © 1995 John Wiley & Sons, Inc.     

Anaerobic formation of succinate from glucose and bicarbonate in resting cells of Leishmania donovani

The cell suspension of Leishmania donovani incorporates ¹⁴CO₂ resulting in the formation of [¹⁴C]-succinic acid under anaerobic conditions. The results showed that the [¹⁴C]-succinate formation from [1-¹⁴C]-glucose is much greater than that from [6-¹⁴C]-glucose. [¹⁴C-pyruvate] takes part in the production of succinic acid under anaerobic conditions without decarboxylation. The anaerobic formation of succinate appears to involve the production of malate, which is then converted to succinate via the reduction of fumarate by the reversal of the tricarboxylic acid cycle. Evidence indicated that the active species in this carboxylation reaction was CO₂ although HCO₃ was active to some extent.     

Mechanism for electric breakdown in a chemically nonequilibrium system and the influence of the chain oxidation reaction in an H2-air mixture on the breakdown threshold

A mathematical model is developed and a numerical analysis is performed for an electric breakdown in a hydrogen-air mixture with a low concentration of H₂. It is shown that, at sufficiently low pressures p<10^?2 atm, a small molecular-hydrogen additive (η=5×10^?5–5×10^?3) decreases the reduced field of an electric breakdown in air by a factor of more than 2 because of the appearance of an additional detachment process associated with the chain hydrogen-oxidation reaction. Detailed calculations are performed for the mean number density of negative oxygen ions [O ₂ ^? ]=10³ cm^?3 and the hydrogen concentration in air [H₂]=0.5, 0.05, and 0.005%. It is found that, for [H₂]=0.005%, the breakdown can develop under the action of a geoelectric field of 1.3 V/cm at p?10^?4 atm.     

RELATIONSHIP BETWEEN PHOTOSYNTHESIS AND CELL SIZE OF MARINE DIATOMS12

Three photosynthetic parameters of 7 species of marine diatoms were studied using Na₂¹⁴CO₃ at 5–8 C using log phase axenic cultures. The cell volumes of the different species varied from 70 μm³ to 40 × 10⁵μm³. The present experiment is consistent with the interpretation that the initial slope α (mg C · [mg chl a]^?1· h^?1· w^?1· m²) of photosynthesis vs. light curves is controlled by self-shading of chlorophyll a in the cell. Pm, the rate of photosynthesis at light saturation (mg C · [mg cell, C]^?1· h^?1) and R, the intercept at zero light intensity (mg C · [mg cell C]^?1· H^?1) are both dependent on the ratio of surface area to volume of cell.     

Isolation and characterization of extracellular glucose oxidase from Penicillium adametzii LF F-2044.1

Hydroxides of magnesium and zinc, aluminum oxide, zinc phosphate, and co-precipitated Ca₃(PO₄)₂ and Mg(OH)₂ were efficient in binding extracellular glucose oxidase (GO) of P. adametzii LF F-2044.1 in a culture liquid filtrate (CLF). Basic Al₂O₃ was the most appropriate adsorbent for GO isolation from the CLF of the fungus. A GO isolation method was developed, which allowed for obtaining an enzyme with a high degree of purification. Spectral properties of the enzyme, its catalytic activity, and stability were characterized. The GO of P. adametzii LF F-2044.1 exhibited high pH stability, retaining activity within the range 4.5–9.0. The rate that GO-catalyzed D-glucose oxidation increased as the temperature increased (up to approximately 60°C). The catalytic activity and thermal stability of GO depended on its concentration in the medium. Under optimum conditions, the fractions GO-1 and GO-2 were characterized by K _M values of 1.56 × 10^?2 and 2.19 × 10^?2 M, respectively; the corresponding values of k _cat equaled 235.1 and 318.2 s^?1.     

Chemiluminescence intensities and spectra of luminol oxidation by sodium hypochlorite in the presence of hydrogen peroxide

Hydrogen peroxide amplifies the chemiluminescence in the oxidation of luminol by sodium hypochlorite. A linear relationship between concentration of hydrogen peroxide and light intensity was found in the concentration range 5 × 10^?8?7.5 × 10^?6 mol/l. At 7.5 × 10^?6 mol/l H₂O₂ the chemiluminescence is amplified 550—fold. The chemiluminescence spectra of these reactions have a wavelength maximum at 431 nm independent of the concentration of hydrogen peroxide. The results indicate that hydrogen peroxide is a necessary component in the chemiluminescent oxidation of the luminol by sodium hypochlorite.