A Biochemical Investigation into the Inhibitory Effects of Glucosamine and N-Acetylglucosamine on the Aggregation In Vitro of Embryonic Chick Muscle Cells

1971 ◽  
Vol 9 (1) ◽  
pp. 85-101
Author(s):  
C. W. LLOYD ◽  
R. B. KEMP
Keyword(s):  
Lactic Acid ◽  
Oxygen Uptake ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Muscle Cells ◽  
Cell Aggregation ◽  
Experimental Period ◽  
Metabolic Effects ◽  
In Cells

The effect of glucosamine and N-acetylglucosamine on aggregation and energy metabolism was investigated over an 8-h period in cells dissociated by 0.25% (w/v) trypsin from the skeletal muscle of 9-day-old chick embryos. At 8 h, 0.023 M glucosamine and N-acetylglucosamine inhibited the aggregation of cells suspended in Eagle's minimal essential medium by 18.9% and 16.4% respectively, as judged on a basis of aggregate size. Glucosamine and N-acetylglucosamine reduced the cellular ATP level by a mean of 36% and 27% respectively; values reflected in the 32% and 19% loss of total adenine nucleotides caused by these sugars. The adenine nucleotide balance was also changed from a mean control ATP/AMP ratio of 13.7 to 8.75 by N-acetylglucosamine and to 8.0 by glucosamine. Intracellular lactate/pyruvate ratios were similarly disrupted in cells incubated with 0.023 M hexosamine. Although the hourly values fluctuated, it was seen that the amount of lactic acid relative to pyruvic acid, considered as an average for the 8-h period, was raised from 6:1 in controls to 8:1 in N-acetylglucosamine-treated and to 10:1 in glucosamine-treated cell preparations. Compared to controls at 8 h, glucosamine enhanced the production of lactate into the suspension medium by 99%. The N-acetyl analogue caused cells to produce more lactic acid than did controls for 4-5 h only, for by 8 h 25% less of this metabolite was assayed in the culture medium. The incorporation of D-[U-14C]glucose into glycogen paralleled the results of extracellular lactic acid assays. N-acetylglucosamine inhibited the incorporation by 30% at 4 h, although by 6 h, and for the remainder of the experimental period, there was more 14C-labelled glycogen in these cells than in controls. By contrast, glucosamine inhibited the incorporation of radioactive glucose into glycogen by 42% at 4 h and, unlike N-acetylglucosamine, consistently thereafter. Glucosamine also enhanced cellular oxygen uptake throughout the experimental period, to the extent of 59% at 8 h. The oxygen uptake of N-acetylglucosamine-treated cells was similar to controls until about the 5th hour, when there was a subsequent inhibition which had accumulated to 13% by the end of the experiment. The release of 14CO2 by cells was inhibited by glucosamine. This hexosamine depressed production by 19% at 12 h whereas N-acetylglucosamine inhibited this evolution by 9% at this time. The metabolic effects of these hexosamines on chick muscle cells in vitro are mainly attributed to a central alteration of the adenine nucleotide balance although certain other documented effects of glucosamine are considered to be involved. An inhibition of cell aggregation by glucosamine and N-acetylglucosamine is discussed in terms of a depressed cellular metabolic economy.

scholarly journals Labeling of the releasable adenine nucleotides of washed human platelets

Blood ◽  
1977 ◽  
Vol 49 (1) ◽  
pp. 89-99 ◽  
Author(s):  
HJ Reimers ◽  
MA Packham ◽  
JF Mustard
Keyword(s):  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Energy Charge ◽  
Human Platelets ◽  
Transport Processes ◽  
Adenylate Energy Charge ◽  
Prolonged Incubation ◽  
Atp Pool ◽  
Adenine Nucleotide Pool

Abstract In rabbit platelets, the metabolically active ATP pool equilibrates with the releasable ATP pool within 1 day. The studies showing this have now been extended to human platelets. Human platelets labeled with 14C-adenosine or 14C-adenine were incubated for up to 10 hr in vitro at 37 degrees C. After 10 hr, about 12% of the total platelet 14C-ATP and 14C-ADP had become releasable with thrombin (4.2 units/ml). Lysis of platelets did not occur, since less than 1% of the platelet-bound 51Cr from platelets labeled with this radioisotope appeared in the ambient fluid upon thrombin treatment. The 14C-ATP/14C-ADP ratio of the released adenine nucleotides (7.6) was similar to the 14C-ATP/14C-ADP ratio of the nonreleasable adenine nucleotides (7.1) 2 hr after the labeling with 14C-adenosine. However, upon prolonged incubation (10 hr) in vitro, the 14C-ATP/14C-ADP ratio of the releasable adenine nucleotides decreased to 2.7. The adenylate energy charge and the 14C- ATP/14C-ADP ratio of the metabolic adenine nucleotide pool did not change significantly during the time of observation. The 14C-ATP content of the platelets decreased by less than 1% hr of incubation at 37 degrees C. These observations are interpreted to mean that the 14C is transferred from the metabolically active, nonreleasable adenine nucleotide pool of human platelets into the releasable adenine nucleotide pool as ATP and is partially hydrolyzed there to yield ADP. The transfer of ATP across the storage organelle membrane of platelets may be similar to transport processes in the chromaffin cells of the adrenal medulla and may represent a general phenomenon in cells that possess storage organelles containing adenine nucleotides.

Studies on the Mechanism Underlying the Inhibition by Puromycin of Cell Aggregation In Vitro

1970 ◽  
Vol 7 (2) ◽  
pp. 557-573
Author(s):  
M. J. DUNN ◽  
E. OWEN ◽  
R. B. KEMP
Keyword(s):  
Carbon Dioxide ◽  
Protein Synthesis ◽  
Cellular Metabolism ◽  
Cell Aggregation ◽  
Experimental Period ◽  
Inhibitory Effect ◽  
Dissociated Cells ◽  
Gyratory Shaker ◽  
Reduced Rate

Cells dissociated with 0.25% crude trypsin from the muscle tissue of 9-day-old chick embryos were employed to investigate the effect of puromycin on cellular metabolism. Parallel studies were also made, using the gyratory shaker, to confirm the effectiveness of puromycin in inhibiting cell aggregation and protein synthesis. Puromycin when introduced at a concentration of 10µg/ml into a suspension of cells in Eagle's MEM did not completely inhibit cell aggregation. Small aggregates were formed in the first 4 h of the experiment. Protein synthesis of the rotated cells, as measured by the incorporation of L-[α-14C]leucine into proteins, was arrested by 91.7% within 15 min of introducing puromycin into a cell suspension. The antibiotic retained its inhibitory effect on protein synthesis for the 24-h period of rotation. Puromycin inhibited the cellular oxygen uptake and carbon dioxide evolution of the rotated cells by 40% within 4 h of its introduction. However, treated cells were still respiring, though at a much reduced rate, at the end of the 24-h experimental period. The release of radioactive carbon dioxide by puromycin-treated cells was also inhibited by 40% at the 4-h stage but after 8 h no further 14CO2 was evolved. The presence of the antibiotic markedly inhibited the uptake of glucose by trypsin-dissociated cells. The level of glycogen and lactate in cells suspended in Eagle's MEM was reduced very considerably over a 24-h period. The presence of puromycin accelerated glycogen utilization over the first 6 h of rotation but at 24 h there was a difference of only 0.6% between the glycogen content of treated cells and controls. At 24 h 11.3% less lactate remained in the puromycin-treated cells than in the controls. The ATP/ADP ratio of trypsin-dissociated cells decreased from an initial value of 2.59 to 1.45 after rotation for 24 h. In the presence of puromycin the ATP/ADP ratio was 0.62 at 4 h and had further declined to 0.48 by 24 h. The effects of puromycin on the aggregation, protein synthesis and cellular metabolism of trypsin-dissociated cells are discussed in relation to cellular adhesive mechanisms.

The Effect of Neuraminidase (3:2:1:18) on the Aggregation of Cells Dissociated from Embryonic Chick Muscle Tissue

1970 ◽  
Vol 6 (3) ◽  
pp. 751-766
Author(s):  
R. B. KEMP
Keyword(s):  
Cell Surface ◽  
Muscle Tissue ◽  
Muscle Cells ◽  
Cell Aggregation ◽  
Inhibitory Effect ◽  
Sialic Acids ◽  
Embryonic Chick ◽  
Experimental Conditions ◽  
Cell Counts

Embryonic chick muscle cells were used to investigate the effect of removing cell-surface sialic acids on cell aggregation in vitro. Single cell suspensions were prepared by dissociating skeletal muscle tissue of 9-day-old chick embryos with either crystalline or crude trypsin. Cell aggregation was quantitatively estimated by turbidimetric and gyratory shaker methods. Cells dissociated with crude trypsin and suspended in Hanks's balanced salts solution (BSS) containing 25u./ml neuraminidase (NANase) only aggregated for 2h when rotated in an absorptiometer. The inhibitory effect of the enzyme was more pronounced with increasing concentration up to 25u./ml. Cells dissociated with crystalline trypsin and treated with 100u./ml NANase immediately exhibited a reduced aggregative competence when gyrated in Eagle's minimum essential medium (MEM) containing 25u./ml NANase, compared with the controls which were not exposed to NANase. The aggregation rate of muscle cells pretreated with 100u./ml NANase and suspended in Eagle's MEM was similar to that of the untreated controls. Cell counts showed that under all three experimental conditions cells were not added to aggregates after the 12-h stage. Aggregates formed in Eagle's MEM (the controls) joined together to form larger aggregates after 12 h, but those rotating in the presence of NANase did not display this property. Lissamine green viability tests showed that cells remained alive throughout the 24-h period in the presence of NANase. Determinations of oxygen uptake, protein synthesis and mitotic index confirmed that general cellular viability was not affected by NANase. Fluorescent-labelled NANase was not taken up by the cells. Treatment of crystalline trypsin-dissociated muscle cells with 100u./ml NANase for 30 min at 37°C significantly reduced their negative electrophoretic mobility. This diminution closely corresponded to the removal of cell-surface sialic acids, as measured by colorimetric tests. Interpretation of the results in the light of current theories of cell adhesion failed to give support to the concept of adhesion by physical forces. The mechanism by which cellular deformability could influence cellular adhesiveness is modified in the knowledge of the present results.

Hepatotrophic effects of insulin on glucose, glycogen, and adenine nucleotides in hepatocytes isolated from fed adult rats

1980 ◽  
Vol 58 (10) ◽  
pp. 1004-1011 ◽  
Author(s):  
Khursheed N. Jeejeebhoy ◽  
Joseph Ho ◽  
Rajni Mehra ◽  
Alan Bruce-Robertson
Keyword(s):  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Energy Charge ◽  
Close Correlation ◽  
Adult Rats ◽  
Fed State ◽  
Adenine Nucleotide Pool ◽  
Intracellular Glucose

In vivo observations have suggested that there is an hepatotrophic effect of insulin. By contrast, subsequent in vitro work, using the isolated perfused liver system, showed no effect or indeterminate effects of insulin on the transport of glucose into the hepatocyte. However because this system may not have endured long enough to show such an influence we explored the transport of glucose using a 48-h suspension culture of hepatocytes isolated from young adult fed rats, the suspension being infused continuously with insulin at a rate approximating the maximum entering portal blood in the fed state. (In a separate study phloridzin was added after 2 h of incubation.) DNA, intracellular glucose and its inward transport, glycogen, and the adenine nucleotides were measured at intervals. By comparison with control or untreated cells, insulin-treated cells showed significantly more DNA and intracellular glucose, and the differences were abolished by phloridzin. Glucose transport rates fell to low values in untreated controls and still lower with insulin plus phloridzin. but the initial rate was maintained to the end (48 h) by insulin alone. Results for glycogen were similar to those for intracellular glucose. There was a close correlation (r = 0.96) between these two. The total adenine nucleotide pool and the concentration of ATP were maintained for about 24 h and fell to half their initial values by 48 h. Insulin had increased these concentrations significantly by 6 h. Although concentrations of ADP and AMP decreased gradually in all groups of cells, insulin enhanced the level of ADP by 12 h but had no measurable effect on that of AMP. The energy charge increased slightly throughout incubation but more so (by 6 h) in the presence of insulin. In conclusion the data support the concept that in the longer term (> 12 h) insulin in the portal circulation maintains the characteristic free permeability of the hepatocyte to glucose and this permits a variety of effects related to glucose entry into the hepatocyte.

Modulation of smooth muscle phenotype in vitro by homologous cell substrate

2003 ◽  
Vol 284 (6) ◽  
pp. C1531-C1541 ◽  
Author(s):  
F. Tao ◽  
S. Chaudry ◽  
B. Tolloczko ◽  
J. G. Martin ◽  
S. M. Kelly
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Cell Phenotype ◽  
Airway Smooth Muscle Cells ◽  
Cell Substrate ◽  
Intracellular Ca ◽  
As Stress ◽  
In Cells

We have developed a novel cell culture system that supports the shortening of smooth muscle cells. Primary rat airway smooth muscle cells were plated on an ethanol-fixed, confluent monolayer of homologous smooth muscle cells (homologous cell substrate, HCS). Cells grown on HCS exhibited morphological and functional characteristics consistent with a differentiated phenotype. Cells on HCS were spindle shaped with a well-defined long axis, whereas cells grown on glass were larger and irregularly shaped. Smooth muscle-specific α-actin immunostained diffusely in cells on HCS, whereas it appeared as stress fibers in cells on glass. Agonists recruited a greater fraction of HCS cells to contract, resulting in greater changes in cell area or length on average, but the maximal capacity of shortening of individual cells was similar between the groups. Unlike cells on glass, cells on HCS shortened to methacholine. HCS was reversible and persisted over several passages. Agonists stimulated intracellular Ca2+ oscillations in cells on HCS, whereas they elicited biphasic peak and plateau transients in cells on glass. HCS modulates smooth muscle cell phenotype in vitro.

scholarly journals Decrease in subunit aggregation of phosphoribosylpyrophosphate synthetase: a mechanism for decreased nucleotide concentrations in pyruvate kinase-deficient human erythrocytes

Blood ◽  
1986 ◽  
Vol 68 (5) ◽  
pp. 1024-1029
Author(s):  
CR Zerez ◽  
NA Lachant ◽  
KR Tanaka
Keyword(s):  
Pyruvate Kinase ◽  
Nicotinamide Adenine Dinucleotide ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Gel Permeation Chromatography ◽  
Metabolic Abnormalities ◽  
Gel Permeation ◽  
Reduced Nicotinamide Adenine Dinucleotide

Pyruvate kinase (PK)-deficient RBCs have several unexplained metabolic abnormalities, such as decreased concentrations of total adenine nucleotides (AMP, ADP, and ATP) and total (oxidized and reduced) nicotinamide adenine dinucleotide (NAD). Because 5-phosphoribosyl-1- pyrophosphate (PRPP) is an intermediate in the synthesis of adenine nucleotides and NAD, we investigated PRPP synthetase (PRPPS), the enzyme responsible for PRPP synthesis. This enzyme is regulated, in part, by changes in its state of subunit aggregation. The proportion of aggregated PRPPS can be altered in vitro by ATP and 2,3- diphosphoglycerate (DPG). Because PK-deficient RBCs have decreased ATP and increased DPG concentrations, we examined the state of subunit aggregation of PRPPS in RBCs from normal and PK-deficient subjects, using gel permeation chromatography. Young normal RBCs have more aggregated PRPPS than do older RBCs. In contrast, due to their decreased ATP and increased DPG concentrations, PK-deficient RBCs contain less aggregated PRPPS than do RBCs of comparable age without PK deficiency. These data suggest that PRPPS should be less active in vivo in PK-deficient RBCs. This may play a key role in mediating the decreases in total adenine nucleotide and total NAD concentrations in these RBCs.

scholarly journals Decrease in subunit aggregation of phosphoribosylpyrophosphate synthetase: a mechanism for decreased nucleotide concentrations in pyruvate kinase-deficient human erythrocytes

Blood ◽  
1986 ◽  
Vol 68 (5) ◽  
pp. 1024-1029 ◽  
Author(s):  
CR Zerez ◽  
NA Lachant ◽  
KR Tanaka
Keyword(s):  
Pyruvate Kinase ◽  
Nicotinamide Adenine Dinucleotide ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Gel Permeation Chromatography ◽  
Metabolic Abnormalities ◽  
Gel Permeation ◽  
Reduced Nicotinamide Adenine Dinucleotide

Abstract Pyruvate kinase (PK)-deficient RBCs have several unexplained metabolic abnormalities, such as decreased concentrations of total adenine nucleotides (AMP, ADP, and ATP) and total (oxidized and reduced) nicotinamide adenine dinucleotide (NAD). Because 5-phosphoribosyl-1- pyrophosphate (PRPP) is an intermediate in the synthesis of adenine nucleotides and NAD, we investigated PRPP synthetase (PRPPS), the enzyme responsible for PRPP synthesis. This enzyme is regulated, in part, by changes in its state of subunit aggregation. The proportion of aggregated PRPPS can be altered in vitro by ATP and 2,3- diphosphoglycerate (DPG). Because PK-deficient RBCs have decreased ATP and increased DPG concentrations, we examined the state of subunit aggregation of PRPPS in RBCs from normal and PK-deficient subjects, using gel permeation chromatography. Young normal RBCs have more aggregated PRPPS than do older RBCs. In contrast, due to their decreased ATP and increased DPG concentrations, PK-deficient RBCs contain less aggregated PRPPS than do RBCs of comparable age without PK deficiency. These data suggest that PRPPS should be less active in vivo in PK-deficient RBCs. This may play a key role in mediating the decreases in total adenine nucleotide and total NAD concentrations in these RBCs.

scholarly journals Labeling of the releasable adenine nucleotides of washed human platelets

Blood ◽  
1977 ◽  
Vol 49 (1) ◽  
pp. 89-99 ◽  
Author(s):  
HJ Reimers ◽  
MA Packham ◽  
JF Mustard
Keyword(s):  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Energy Charge ◽  
Human Platelets ◽  
Transport Processes ◽  
Adenylate Energy Charge ◽  
Prolonged Incubation ◽  
Atp Pool ◽  
Adenine Nucleotide Pool

In rabbit platelets, the metabolically active ATP pool equilibrates with the releasable ATP pool within 1 day. The studies showing this have now been extended to human platelets. Human platelets labeled with 14C-adenosine or 14C-adenine were incubated for up to 10 hr in vitro at 37 degrees C. After 10 hr, about 12% of the total platelet 14C-ATP and 14C-ADP had become releasable with thrombin (4.2 units/ml). Lysis of platelets did not occur, since less than 1% of the platelet-bound 51Cr from platelets labeled with this radioisotope appeared in the ambient fluid upon thrombin treatment. The 14C-ATP/14C-ADP ratio of the released adenine nucleotides (7.6) was similar to the 14C-ATP/14C-ADP ratio of the nonreleasable adenine nucleotides (7.1) 2 hr after the labeling with 14C-adenosine. However, upon prolonged incubation (10 hr) in vitro, the 14C-ATP/14C-ADP ratio of the releasable adenine nucleotides decreased to 2.7. The adenylate energy charge and the 14C- ATP/14C-ADP ratio of the metabolic adenine nucleotide pool did not change significantly during the time of observation. The 14C-ATP content of the platelets decreased by less than 1% hr of incubation at 37 degrees C. These observations are interpreted to mean that the 14C is transferred from the metabolically active, nonreleasable adenine nucleotide pool of human platelets into the releasable adenine nucleotide pool as ATP and is partially hydrolyzed there to yield ADP. The transfer of ATP across the storage organelle membrane of platelets may be similar to transport processes in the chromaffin cells of the adrenal medulla and may represent a general phenomenon in cells that possess storage organelles containing adenine nucleotides.

In vivo control of phosphofructokinase: system models suggest new experimental protocols

1989 ◽  
Vol 257 (4) ◽  
pp. R878-R888 ◽  
Author(s):  
R. J. Connett
Keyword(s):  
Adenine Nucleotide ◽  
Energy State ◽  
Adenine Nucleotides ◽  
Experimental Studies ◽  
Energy System ◽  
Absolute Size ◽  
Steady State Kinetics ◽  
Adenine Nucleotide Pool

There is still uncertainty as to how much control of in vivo rates of glycolysis by phosphofructokinase (PFK) depends on cytosolic phosphate energy state. Three models of PFK kinetics incorporating sensitivity to pH, adenine nucleotides, and inorganic phosphate (Pi) were embedded in the physiological "phosphate energy system" of creatine-containing tissues [Connett, R.J. Am. J. Physiol. 254 (Regulatory Integrative Comp. Physiol. 23): R949-R959, 1988]. Effects of changes in phosphate energy state and total adenine nucleotide and phosphate pools on steady-state kinetics were examined. Analyses mimicking in vitro experiments indicated no activity at the pH and [ATP] of working muscles. When tested using the coordinated changes in Pi and adenine nucleotides expected in vivo, all models showed reasonable activity. Control was dominated by [Pi] in the normal physiological range of energy states. The almost linear response to phosphate energy state, measured by creatine charge (phosphocreatine/total creatine), is insensitive to the absolute size of the adenine nucleotide pool. A step to almost full activation occurred when phosphocreatine buffering of [ATP] was exceeded. Several experimental studies are suggested.

Malignant hyperthermia: slow sodium current in cultured human muscle cells

1989 ◽  
Vol 257 (4) ◽  
pp. C759-C765 ◽  
Author(s):  
S. J. Wieland ◽  
J. E. Fletcher ◽  
H. Rosenberg ◽  
Q. H. Gong
Keyword(s):  
Malignant Hyperthermia ◽  
Sodium Current ◽  
Muscle Cells ◽  
Slow Inward Current ◽  
Ion Currents ◽  
Inward Current ◽  
Anesthetic Agents ◽  
Lower Magnitude ◽  
In Cells

Voltage-activated ion currents were measured in cultured skeletal muscle myoballs. Cultures were generated from biopsies from patients referred for diagnosis of susceptibility to malignant hyperthermia (MH); diagnosis of susceptibility (MH+) or nonsusceptibility (MH-) was made on the basis of in vitro halothane-induced contracture of a separate piece of biopsy. Measurements of ion currents were made at room temperature in the absence of anesthetic agents, using tight-seal whole-cell recording. Fast transient Na+ currents and delayed outward K+ currents were similar in magnitude and kinetics in cells from MH+ and MH- patients. An additional slowly inactivating inward current component was commonly observed in cells from MH+ patients. This current was blockable by tetrodotoxin and was carried by Na+ but not by Ba2+. The component was less frequently observed and was of a lower magnitude in cells from MH- patients. The increased magnitude of the slow inward current observed in cultured muscle cells from MH+ patients may be a manifestation of the lesion that causes MH.

Sign in / Sign up

Export Citation Format

Share Document