Differential activation of membrane phospholipid turnover by compound 48/80 and ionophore A23187 in rat mast cells

Membrane phospholipid turnover was investigated during histamine release from rat mast cells. Addition of calcium ionophore A23187 (0.5 microgram/ml) to mast cells prelabeled with [3H]glycerol induced the rapid and progressive increase in phosphatidic acid (PA) and 1,2-diacylglycerol (DG), which was concomitant with the small rise in phosphatidylinositol (PI). Loss of the level in triacylglycerol (TG) was very marked. Polyamine compound 48/80 (5 micrograms/ml) was shown to cause rises in PA, 1,2-DG, and PI without any significant changes in TG. Both stimuli increased incorporation of exogenous [3H]glycerol into phospholipids, indicating the involvement of de novo synthesis in phospholipid metabolism. Studies with [3H]arachidonic acid-labeled mast cells showed an enhanced liberation of radioactive arachidonate and metabolites upon histamine release. There were associated decreases of radioactivity in phosphatidylcholine (PC) and TG when exposed to A23187, while phosphatidylethanolamine (PE) was degraded as a result of 48/80 activation. The transient increases of [3H]arachidonoyl-1,2-DG and PA were caused by 48/80, while A23187 showed a gradual rise in the radioactivity in these two lipid fractions. These findings reflect activation of phospholipase C. When mast cells were activated by low concentrations of A23187 (0.1 microgram/ml) and 48/80 (0.5 microgram/ml), different behaviors of PI metabolism were observed. An early degradation of PI and a subsequent formation of 1,2-DG and PA suggest that the lower concentrations of these agents stimulate the PI cycle initiated by PI breakdown rather than de novo synthesis. These results demonstrate that marked and selective changes in membrane phospholipid metabolism occur during histamine release from mast cells, and that these reactions seem to be controlled by the coordination of degradation and biosynthesis, depending on the type and the concentration of stimulants. A23187 stimulates arachidonate release perhaps via the cleavages of PC and TG, whereas 48/80 liberates arachidonate from PE.     

The effect of ionophore A23187 on albumin internalization in cultured human umbilical vein endothelial cells

The effects of calcium and the calcium ionophore A23187 on endocytosis were studied in cultured human umbilical vein endothelial cells using iodinated human albumin to measure bulk phase endocytosis. In the absence of the ionophore, varying the levels of extracellular calcium did not affect endocytosis. In the presence of 10 μM A23187, the endocytic clearance of albumin decreased approx. 50% when exposed to physiological concentrations of extracellular calcium, but increased approx. 50% at lower calcium concentrations. Since the ionophore is known to alter cellular calcium levels, these results are compatible with a role for intracellular calcium in the modulation of endothelial cell endocytosis.     

Effects of pertussis vaccine on the lipid metabolism of hamsters

Administration of pertussis vaccine to hamsters markedly affected their lipid metabolism. Four days after the administration of the vaccine a severe fatty liver was observed. Concomitantly, a rise in the serum levels of free fatty acids, triacylglycerols and ketone bodies was detected. It is suggested that an altered regulation of adipose tissue lipolysis might be at least partially responsible for the observed effects.     

Enhanced insulin stimulation of sugar transport and DNA synthesis by glucocorticoids in cultured human skin fibroblasts

Glucocorticoids will enhance the growth of cultured human skin fibroblasts in serum-containing medium. In serum-free cultures hydrocortisone (5 X 10(-6) M) will enhance insulin stimulation of sugar transport and DNA synthesis (as measured by thymidine incorporation into trichloroacetic acid-precipitable material). The optimal concentration for the glucocorticoid effect on DNA synthesis was 5 X 10(-8) M for dexamethasone and 5 X 10(-7) M for hydrocortisone. In dexamethasone-treated cells, concentrations of insulin as low as 250 microU/ml (10 ng/ml) were effective in stimulating DNA synthesis. Further, hydrocortisone and dexamethasone (both at 5 X 10(-6) M) exhibited potentiating effects on insulin-stimulated sugar transport. These effects appeared to be mediated via inhibitory actions on the hexose transport system with the preservation of a functional insulin-receptor interaction resulting in insulin stimulation of deoxy-D-glucose transport at physiological insulin concentrations, 250 microU/ml (10 ng/ml). Hydrocortisone also enhanced specific [125I]insulin binding in these cells. The data indicate that the mechanism(s) of glucocorticoid enhancement of two actions of insulin may be different.     

Regulation of insulin binding and stimulation of sugar transport in cultured human fibroblasts by sugar levels in the culture medium

Studies were carried out on cultures of human skin fibroblasts to explore the effects of culture medium glucose levels on insulin binding and action. Cell cultures in 5.55 mm glucose-containing medium depleted their medium glucose within 3 days, and at that time exhibited elevated deoxy-d-glucose (2-DG) transport (84% greater than control cultures fed 22.2 mm glucose) and failure of insulin to stimulate 2-DG transport (an insulin:control transport ratio of 1.02). There was also a significant negative correlation between basal 2-DG transport and insulin binding (r = ?0.621; n = 29; P < 0.01), while insulin binding exhibited a significant positive correlation with insulin action (r = 0.816; n = 12; P < 0.01). Glucose starvation of cultures for 18 h resulted in several changes: (i) a 49% decrease in specific ¹²⁵I-insulin binding due to a reduction in binding capacity; (ii) elevated basal 2-DG transport; and (iii) an absence of insulin stimulation of 2-DG transport. Exposure to increasing concentrations of glucose for 18 h led to a glucose concentration-dependent increase in specific insulin binding. Additionally, the various changes in the glucose-starved group were reversed after as little as 6 h of glucose refeeding. The results indicate that basal sugar transport, and insulin binding and action can be regulated by the amount of glucose in the medium.     

Stereospecific binding of 3H-phencyclidine in brain membranes

Phencyclidine (PCP) displaceable binding of ³H-PCP to glass-fiber filters was eliminated and total binding markedly reduced by initial treatment of the discs with 0.05% polyethyleneimine. Assessed with treated filters, unlabeled PCP displaced ³H-PCP in both rat and pigeon brain membranes with an EC50 of 1 μM. Of similar high inhibitory potency were dextrorphan, levorphanol, SKF 10047 and ketamine, while morphine, naloxone and etorphine had EC50 values higher then 1 mM. Using the dissociative anesthetic dexoxadrol and its inactive isomer levoxadrol as displacing agents, stereospecific binding of ³H-PCP was obtained in rat and pigeon brain membranes. The markedly higher potency of dexoxadrol, relative to levoxadrol, in displacing bound ³H-PCP is compatible with behavioral data for these enantiomers. However, they were equipotent in displacing ³H-PCP bound to glass-fiber filters in the absence of tissue. Heat denaturation, but not freezing, abolished stereospecific binding of ³H-PCP, which was also absent in rat liver membranes. The stereospecific binding component in brain displayed biphasic saturability at 60–70 nM and 300–400 nM, respectively.     

Effects of chronic ethanol feeding and acetaldehyde metabolism on calcium transport by rat liver mitochondria

The prolonged feeding of ethanol to rats alters in vitro mitochondrial transport of calcium. Hepatic mitochondria isolated from rats fed ethanol for 7 weeks exhibited decreased retention of calcium in the presence of 4mM-Pi. This defect was associated with enhanced efflux of calcium when mitochondria were incubated with EGTA. Acetaldehyde at low, "physiological" concentrations (100 microM) enhanced calcium retention by mitochondria but this response was blunted after chronic ethanol administration. The in vitro actions of acetaldehyde appear to be mediated, in part, by its metabolism in mitochondria since pretreatment of rats with cyanamide (an aldehyde dehydrogenase inhibitor) prevents this effect.     

A comparison of the uptake, metabolism, and action of cyclic adenine nucleotides in cultured hepatoma cells.

Intracellular radioactivity following incubation of HTC or RLC cells in [³H]cAMP exceeds that following incubation in either [³H]mono- or dibutyryl cAMP by 30-fold, yet little [³H]cAMP is found within the cells. Even at early times (30 min) the label derived from [³H]cAMP is predominantly found in ADP or ATP, suggesting it mostly enters the cell as the nucleoside. Significant intracellular concentrations of monobutyryl cAMP (2–10 μm) result from incubation of both cell lines in either N⁶ mono- or dibutyryl cAMP. A very small percentage of this label is in cAMP, and within 2 h of incubation > 65% of the label is again found in ADP or ATP.Liver cytosol contains three major cAMP-dependent protein kinases, designated A, B, and C, as resolved by DEAE-Sephadex chromatography. cAMP is the most effective in vitro activator (10- to 16-fold stimulation) of kinases A and B, the preponderant forms, in the order cAMP > N⁶ monobutyryl cAMP ? dibutyryl cAMP. Kinase C, a minor fraction, was stimulated two to threefold with the order cAMP ≥ N⁶ monobutyryl cAMP > dibutyryl cAMP. HTC and RLC cell cytosol protein kinase has Chromatographic and cyclic nucleotide activation properties similar to those of liver fraction C.The activation state of the protein kinases of HTC and RLC cells incubated in the various cyclic nucleotides was also studied. The ability of such nucleotides to occupy regulatory protein binding sites in intact cells (as determined by the inhibition of subsequent in vitro binding of [³H]cAMP) was of the order N⁶ monobutyryl cAMP > dibutyryl cAMP > cAMP > untreated cells. Correspondingly, the ratio of basal protein kinase activity in cyclic nucleotide treated:control cells was higher in cells incubated in monobutyryl cAMP > dibutyryl cAMP > cAMP. This in vivo activation suggests that little additional stimulation would be obtained by adding cAMP to extracts prepared from such cells. This activation can be expressed as the ratio ? cAMP: + cAMP (a ratio of 1 being maximal activation). The highest such ratio was seen in cells which had been incubated in monobutyryl cAMP > dibutyryl cAMP > cAMP > untreated cells. The studies indicate that all three cyclic nucleotides are capable of activating protein kinase in intact RLC and HTC cells; however the monobutyryl derivative is the most effective, and the degree of stimulation is greater in RLC than in HTC cells.RLC cell tyrosine aminotransferase activity is increased two to threefold by butyrylated cAMP derivatives (but not by cAMP) whereas the HTC cell enzyme is not induced. The rate of replication of both lines is unaltered by the butyrylated compounds.Since HTC and RLC cells accumulate and metabolize cAMP and its derivatives equally, and since they both contain a protein kinase with similar in vivo and in vitro activation properties, it is suggested that the effects of butyrylated cAMP derivatives on cell replication and tyrosine aminotransferase induction are mediated separately, either by distinct protein kinases, or at a point distal to protein kinase, or by a mechanism independent of protein kinase.     

An ultramicro method of amino acid analysis: application to studies of protein metabolism in cultured cells.

Analysis of the quantity and specific radioactivity of amino acids derived from intra-cellular pools, aminoacyl-transfer RNA, and protein hydrolysates of cultured cells has been achieved using a radiolabeled amino group ligand, dansyl chloride. Speeific activities of ¹⁴C- or ³H-labeled amino acids are calculated after reaction with appropriately labeled dansyl chloride of known specific activity. The quantity of amino acid is determined as a function of its diluting influence on a radioactive standard. The specific activity of as little as 2 pmol of amino acid can be measured using [¹⁴C]dansyl chloride the less sensitive of the two isotopic species available. Thus, cells from a single 60-mm culture dish provide sufficient material for analysis of both intracellular and transfer RNA amino acid pools, and one can easily analyze the amino acids in hydrolysates made from individual bands in polyacrylamide gels. The method offers significant improvement in speed, sensitivity, and economy over conventional methods of amino acid analysis and, because of its double-label design, gives accurate results with a minimum of technical expertise and no major equipment other than a scintillation counter.     

Effects of atractyloside and palmitoyl coenzyme A on calcium transport in cardiac mitochondria.

Palmitoyl CoA (PCoA) and the adenine translocase inhibitor atractyloside (ATR) appear to produce a similar effect in discharging accumulated calcium from cardiac mitochondria. Although mitochondrial respiration is stimulated upon addition of either PCoA or ATR to preparations preloaded with calcium, the effect is not the same as that produced by classical uncouplers. PCoA and ATR also do not interfere with respiration-supported calcium uptake by mitochondria. The presence of exogenous ATP can prevent the calcium discharging effects of PCoA or ATR. Carnitine will prevent the PCoA calcium discharging effect, but has no effect on ATR-induced discharge. It is suggested the PCoA may act at a site on or near the adenine translocase, perhaps through allosteric interaction, to produce an efflux of calcium from mitochondria. The results also suggest that the internal adenine nucleotide pool plays a significant role in mitochondrial calcium retention.     

Effects of thermodynamic nonideality in kinetic studies

Experimental evidence is presented for concentration dependence of the pseudo-firstorder rate constant describing the rate of inversion of sucrose by 2 m HCl; and also of the increase in maximal velocity for the catalytic reduction of pyruvate by lactate dehydrogenase that results from addition of the inert macromolecular solutes bovine serum albumin, ovalbumin, and Dextran T70. These somewhat unusual and seemingly diverse observations are examined in terms of a theory formulated on the basis of two equilibrium reactions, the first describing complex formation between two reactants, and the second isomerization of that complex to an activated state prior to product formation. This formulation permits consideration of activity coefficient ratios relevant to the equilibria and the expression of these ratios as power series in total solution composition. Quantitative assessment of the experimental results is made possible in these terms by estimating the magnitudes of the constant coefficients of the virial expansions as excluded volumes. It is concluded that the result observed in the sucrose inversion study finds rational explanation in thermodynamic nonideality factors governing the overall equilibrium between the reactants and the activated complex of sucrose and hydronium ion. For the enzyme-catalyzed reaction the same general equation applies but particular attention is given to the simplified form that is relevant to high substrate concentrations, where, in the absence of inert compounds, the conventional maximal velocity is approached. In this region an increase in velocity observed upon addition of an inert macromolecular component may be considered explicitly in terms of excluded volume effects related to a shape change in the isomerization between enzyme-substrate complex and its activated state.     

Effects of calcitonin and parathyroid hormone on the metabolism of chondrocytes in culture

Rabbit articular chondrocytes in suspension culture synthesize Type II colagen [3α₁(II)] in the absence of extracellular Ca²⁺ and Type Icollagen [2α₁?(I)·α₂] in the complete medium. As a result of pre-treatment in monolayer culture with calcitonin or parathyroid hormone in the complete medium, an influx of Ca²⁺ into the cells occurs. These cells produce mainly Type I collagen when transferred to suspension cultures in the medium devoid of CaCl₂. If added directly to the suspension culture medium containing no CaCl₂, calcitonin stimulates an active efflux of Ca²⁺ from the cells into the medium and leads the cells to synthesize Type I collagen. Under similar conditions, parathyroid hormone does not change the collagen-phenotype.     

Synergistic functions of phorbol ester and calcium in serotonin release from human platelets

In human platelets, thrombin activates Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) and mobilizes Ca2+ concomitantly, whereas 12-O-tetradecanoylphorbol-13-acetate (TPA) may be intercalated into membranes and directly activates protein kinase C without mobilization of Ca2+ in sufficient quantities. A series of experiments with TPA and Ca2+-ionophore (A23187) indicates that activation of protein kinase C is a prerequisite requirement for release of serotonin, and that this enzyme activation and Ca2+ mobilization act synergistically to elicit a full cellular response. Both cyclic AMP and cyclic GMP inhibit activation of protein kinase C by prohibiting the signal-dependent breakdown of inositol phospholipid to produce diacyl-glycerol, but none of these cyclic nucleotides prevents the TPA-induced activation of this enzyme.     

Analysis of microtubule polymerization inhibitors in sea urchin egg extracts: Evidence for a protease

Inhibitors of microtubule polymerization have been found in extracts of unfertilized sea urchin eggs using neural tubulin polymerization assays without glycerol. The inhibitory activity is partially destroyed by boiling or by reduction and carboxymethylation and is nondialyzable. When chromatographed on DEAE-cellulose, the inhibitory activity is eluted over a broad NaCl gradient and is in association with several peaks. This partially purified inhibitor is not destroyed by incubation with RNase A. When the partially purified inhibitor is incubated with brain microtubule protein under conditions which support microtubule polymerization, both high molecular weight-microtubule associated proteins and tubulin appear to be digested when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteolytic digestion as well as inhibition of microtubule polymerization depend upon similar concentrations of partially purified inhibitor present in the polymerization reaction. It appears as though at least part of the microtubule polymerization inhibitory activity present in unfertilized sea urchin eggs is due to this protease.     

Effects of cultured astroglia on the survival of neonatal rat retinal ganglion cells in vitro

Retinal ganglion cells (RGC), as identified by retrograde horseradish peroxidase (HRP) labeling technique, were cultured in a minimal medium; only 20% of them survived after 16 hr in vitro. However, superior colliculus-conditioned medium was capable of supporting 100% of RGC over this assay time; enhanced neurite expression also was evident. It was decided to investigate whether glial cells within the superior colliculus may provide a soluble factor capable of supporting RGC. Glial-conditioned medium prepared over monolayers of either predominantly flat astrocytes (relatively immature) or predominantly process-bearing (mature) astrocytes failed to maintain RGC. The possibility that astrocytes may provide support for RGC via membrane contact was then investigated. Dissociated retinae were grown on monolayers consisting primarily of either flat or process-bearing astrocytes. Cultures rich in flat astrocytes maintained over 70% of RGC originally present, and many of them exhibited extensive neurite outgrowth and elongation. Process-bearing astrocytes were unable to support RGC survival. Immature astroglial cells may therefore support RGC via glial-neuronal interaction.     

Distribution of calmodulin in taste buds

Calmodulin is higher in particulate fractions from bovine taste buds containing taste bud membranes which specifically bind sweet tastants compared to corresponding fractions from control non-taste bud bearing lingual epithelial tissue. As biochemical purity (i.e., membrane enzyme marker activity) of these membrane enriched fractions increased (P4B greater than P3B greater than P2B) calmodulin correspondingly increased (P4B greater than P3B greater than P2B); these increases also correlated with increased membrane purity as demonstrated by electron microscopy. All PB subfractions from taste buds contained a greater membrane concentration than those from PD subfractions and calmodulin was significantly increased in each corresponding subfraction. The presence of calmodulin in taste bud membranes, its correlation with membrane purification and reports that numerous drugs which induce taste loss are potent inhibitors of calmodulin suggest a role for calmodulin in taste function.     

Solvent and solute effects on “inside-outside” conformations and rotation about the peptide bond in a miniature protein model

The physicochemical parameters affecting protein unfolding in relation to peptide bond rotations are briefly reviewed. As a suitable model for the study of solvent and solute effects on amide rotation and inside-outside conformations, the 2,2′-biphenyl analog of N-benzoyl-l-phenylalanine methyl ester (I) was synthesized and resolved enzymatically with α-chymotrypsin. The optically pure substance exists as conformer I_a (R,S configuration) with an axial methoxycarbonyl in the crystalline state. Rotation about the biphenyl axis leads to the equatorial conformer I_b (S,S configuration) in various solvents. In polar solvents, rotation about the amide is rate limiting. Accurate measurements of this rotation were accomplished by following the rate of change in the maximum amplitude of the biphenyl Cotton band at 256 nm. The high sensitivity of the method allowed rate and equilibrium measurements at 10^?3M in the absence of intermolecular association. Small differences of the order of 100 cal/mole in ΔG^≠ or ΔG_eq could thus be detected accurately. It was found that k_obs or k₁ (forward step) for equilibration was linearly related (correlation coefficient of 0.96 for k_obs) with E_T, the solvent polarity index on Reichardt and Dimroth's scale. Rotation was slowest in water and fastest in carbon tetrachloride, δΔG^≠, being 2.4 kcal/mole. Chaotropic anions, cations, and guanidinium chloride accelerated the rate in water. However, the inside-outside (axial-equatorial; I_aI_b) ratio at equilibrium did not correlate in any simple manner with the solvent E_T values. Rather, correlation within groups of solvents appeared to exist. It was suggested that solvent association with the amide differs quantitatively in the inside and outside conformations. The position of the equilibrium in water was affected by chaotropic ions but not by urea or quanidinium chloride. Some possible mechanisms are briefly outlined.