scholarly journals Influence of Hormones, Second Messengers and pH on the Expression of Metabolic Responses to Anoxia in a Marine Whelk

1989 ◽  
Vol 145 (1) ◽  
pp. 31-43 ◽  
Author(s):  
STEPHEN P. J. BROOKS ◽  
KENNETH B. STOREY
Keyword(s):  
Pyruvate Kinase ◽  
Second Messengers ◽  
Metabolic Responses ◽  
Kinetic Properties ◽  
Ionophore A23187 ◽  
Ph Change ◽  
Tissue Ph ◽  
Biochemical Responses

The roles of hormones, second messengers and pH in triggering or potentiating biochemical responses to anaerobiosis were evaluated using in vitro incubations of isolated muscle tissues (foot, radular retractor, ventricle) from the marine whelk Busycon canaliculatum (L.). Incubating tissues in vitro under anoxic conditions stimulated changes in muscle fmctose-2,6-bisphosphate levels and pyruvate kinase kinetics (Km values for phosphoenolpyruvate, I50 values for L-alanine) that were virtually equivalent to those that occur in vivo. Additions of hormones (epinephrine, norepinephrine, octopamine, serotonin, glucagon, insulin) or inhibitors of prostaglandin synthesis (dexamethasone, aspirin) had no effect on these metabolic responses to anoxia. The second messenger compounds, dibutyryl cyclic AMP and Ca2+ + ionophore A23187 + phorbol myristate acetate, produced isolated and tissue-specific responses in muscles incubated under aerobic conditions, but the magnitude and pattern of these responses differed from those seen in anoxia. Second messengers also had no effect on the development of biochemical responses in anoxic muscles. Tissue pH was artificially altered in order to evaluate the role of pH change (acidification occurs during anoxia in vivo) in the control of metabolic responses to anoxia. In all cases, the changes in the kinetic properties of pyruvate kinase (PK) correlated with the state of oxygenation of the tissue and not with the measured tissue pH value; higher tissue pH did not prevent anoxiainduced phosphorylation of PK and lower tissue pH did not alter the kinetic patterns of the aerobic enzyme. Overall, the study indicates that cells and tissues of the whelk respond individually to anoxia and that coordination of the action of protein kinases during anoxia is not mediated by pH or by common secondmessenger mechanisms.

Oligomeric Forms Of Pyruvate Kinase From Chlorella With Different Kinetic Properties

1991 ◽  
Vol 46 (5-6) ◽  
pp. 416-422 ◽  
Author(s):  
G. Ruyters ◽  
N. Grotjohann ◽  
W. Kowallik
Keyword(s):  
Pyruvate Kinase ◽  
Total Activity ◽  
Substrate Affinity ◽  
Kinetic Properties ◽  
Resting Cells ◽  
Carbohydrate Degradation ◽  
Enzyme Species ◽  
Oligomeric Forms

Fast protein liquid chromatography on Superose 6 of crude extracts from the chlorophyllfree mutant no. 20 of the unicellular green alga Chlorella kessleri reveals two possibly oligomeric forms of pyruvate kinase (2.7.1.40). Their occurrence is markedly altered in the course of heterotrophic growth with changing levels of exogenous glucose as carbon source with only one enzyme species with a MW of 400 kDa existing in growing cells, two forms of 400 and 580 kDa in resting cells. Substrate affinity towards PEP of the 400 kDa form is better than that of the 580 kDa species; responses to the effector AMP are different as well. In vitro, addition of PEP or of AMP leads to the formation of higher MW enzyme species with MW of 730, 1050 and 1400 kDA without affecting the total activity. In vivo alterations in the levels of several metabolites including PEP upon addition of glucose have been shown to occur. Therefore, it is discussed, whether changes in the concentration of intermediates and effectors may provide the mechanism for the increased rate of carbohydrate degradation by affecting the occurrence and/or ratio of the various PK forms with different kinetic and regulatory properties. Upon blue light irradiation, which also stimulates carbohydrate breakdown of the Chlorella mutant cells, the distribution of PK is shifted towards the species with higher substrate affinity, a result being in accordance with the above conception

scholarly journals Effect of oxalate and malonate on red cell metabolism

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1389-1393
Author(s):  
E Beutler ◽  
L Forman ◽  
C West
Keyword(s):  
Pyruvate Kinase ◽  
Cell Metabolism ◽  
Inhibitory Effect ◽  
Red Cells ◽  
Red Cell ◽  
Acid Analogue ◽  
2,3 Dpg

The addition of oxalate to blood stored in Citrate-phosphate-dextrose (CPD) produces a marked improvement in 2,3-diphosphoglycerate (2,3-DPG) preservation; an increase in 2,3-DPG levels can also be documented in short-term incubation studies. Oxalate is a potent in vitro inhibitor of red cell lactate dehydrogenase, monophosphoglycerate mutase, and pyruvate kinase (PK). In the presence of fructose 1,6-diphosphate the latter inhibitory effect is competitive with phospho(enol)pyruvate (PEP). Determination of the levels of intermediate compounds in red cells incubated with oxalate suggest the presence of inhibition at the PK step, indicating that this is the site of oxalate action. Apparent inhibition at the glyceraldehyde phosphate dehydrogenase step is apparently due to an increase in the NADH/NAD ratio. Oxalate had no effect on the in vivo viability of rabbit red cells stored in CPD preservatives for 21 days. Greater understanding of the toxicity of oxalate is required before it can be considered suitable as a component of preservative media, but appreciation of the mechanism by which it affects 2,3-DPG levels may be important in design of other blood additives. Malonate, the 3-carbon dicarboxylic acid analogue of oxalate late did not inhibit pyruvate kinase nor affect 2,3-DPG levels.

scholarly journals Pattern of pyruvate kinase isozymes in erythroleukemia cell lines and in normal human erythroblasts

Blood ◽  
1984 ◽  
Vol 64 (4) ◽  
pp. 930-936 ◽  
Author(s):  
I Max-Audit ◽  
U Testa ◽  
D Kechemir ◽  
M Titeux ◽  
W Vainchenker ◽  
...  
Keyword(s):  
Pyruvate Kinase ◽  
Cell Lines ◽  
Fetal Liver ◽  
Erythroid Cells ◽  
Low Concentrations ◽  
Erythroleukemia Cell ◽  
Erythroleukemic Cell ◽  
High Level

To further investigate the erythroid nature of the two human erythroleukemia cell lines, K562 and HEL-60, and to define the ontogeny of pyruvate kinase (PK) isozymes (R, M2) in developing human erythroid cells, we have studied the isozymic alterations, if any, during differentiation of these cell lines in vitro and normoblasts isolated from fetal liver in vivo. PK activity of erythroleukemic cell lines was intermediate between that observed in leukocytes and in fetal liver erythroblasts. These cell lines contained a high level of M2-PK, but R- PK was always present, albeit at low concentrations, in all the clones or subclones we studied. Erythroblasts from fetal liver were separated according to density on a Stractan gradient. R-PK levels were nearly constant in the different fractions, whereas M2-PK levels markedly decreased as the erythroblasts became mature and almost completely disappeared in late erythroid cells. Thus, these results clearly demonstrate the erythroid origin of these cell lines.

Bulk isolation of renal PCT and PST. II. Differential responses to anoxia or hypoxia

1990 ◽  
Vol 259 (1) ◽  
pp. F176-F185 ◽  
Author(s):  
C. E. Ruegg ◽  
L. J. Mandel
Keyword(s):  
Lactate Dehydrogenase ◽  
O2 Consumption ◽  
Atp Content ◽  
Hypoxic Injury ◽  
Biochemical Responses ◽  
In Vitro Exposure ◽  
Differential Responses

Innate biochemical responses of rabbit renal proximal convoluted (PCT) and straight (PST) segments following in vitro exposure to anoxia or hypoxia were investigated to delineate the mechanisms responsible for segment-selective injury in vivo. After bulk isolation, suspensions (1 mg/ml) enriched in either PCT or PST were preincubated in Dulbecco's modified Eagle's-Ham's F-12 medium for 1 h before being exposed to either 40 min of anoxia (N2) or 120 min of hypoxia (1% O2) and 1 h of recovery under air-CO2 conditions. After recovery from anoxia, the percent of control values for each viability indicator in PCT and PST, respectively, were as follows: O2 consumption (QO2), 30/50; ATP content, 22/49; K+ content, 60/70; and percent lactate dehydrogenase (LDH) release, 66/45. Likewise, following recovery from hypoxia, the percent of control values for PCT and PST, respectively, were as follows: QO2, 50/90; ATP, 16/57; K+, 52/79; LDH, 45/17. These differential responses indicate that PCT segments were innately more susceptible to anoxic and hypoxic injury than PST segments. Because ATP content was significantly higher in PST segments immediately after anoxia and hypoxia, we investigated glucose-dependent responses during anoxia by exposing these segments to 30 min of anoxia in nutrient buffer with or without glucose. Results from these experiments demonstrate that the PST protection from anoxia was glucose dependent because removal of glucose from the nutrient buffers during anoxia abolishes the differential responses between PCT and PST. The in vitro PCT sensitivity observed here contrasts with the PST sensitivity observed following in vivo ischemia, suggesting that hemodynamic factors present in vivo may ultimately determine the overall susceptibility of PST segments in situ.

scholarly journals Oxygen pressure-dependent control of carbonic anhydrase synthesis in chick embryonic erythrocytes

1991 ◽  
Vol 261 (5) ◽  
pp. R1188-R1196 ◽  
Author(s):  
D. Million ◽  
P. Zillner ◽  
R. Baumann
Keyword(s):  
Carbonic Anhydrase ◽  
Pertussis Toxin ◽  
Second Messengers ◽  
Fold Increase ◽  
Rapid Onset ◽  
Inhibitor Molecule ◽  
Activation Of Transcription ◽  
Plasma Factor

During chick embryonic development carbonic anhydrase (CA) expression of erythrocytes is kept at a very low level until the last week of incubation (i.e., up to day 14). We have previously obtained evidence that hypoxia is the physiological stimulus for rapid onset of CA synthesis before hatching. Looking for putative signals we have carried out in vitro incubations of embryonic erythrocytes, screening a large number of hormones and second messengers, which were all ineffective, with the exception of the A1 agonist N6-phenylisopropyladenosine (adenosine had no effect). However, incubation with embryonic plasma (10%) from embryos greater than 6 days caused a 10-fold increase of the CA activity during 24 h. This increase was not observed when the incubation was carried out with the addition of actinomycin D, cycloheximide, aluminum fluoride, pertussis toxin, or heat-inactivated plasma. Mammalian plasma had no effect on CA activity. Filtration experiments show that the molecular mass of the factor is less than 2,000 Da. We conclude that embryonic plasma contains a heat-labile factor which stimulates CA synthesis via activation of transcription and whose receptor is coupled to a pertussis toxin-sensitive G protein. In vivo the action of the plasma factor is suppressed as long as blood Po2 is high, suggesting the presence of an inhibitor molecule whose synthesis is controlled by the Po2.

scholarly journals Effect of oxalate and malonate on red cell metabolism

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1389-1393 ◽  
Author(s):  
E Beutler ◽  
L Forman ◽  
C West
Keyword(s):  
Pyruvate Kinase ◽  
Cell Metabolism ◽  
Inhibitory Effect ◽  
Red Cells ◽  
Red Cell ◽  
Acid Analogue ◽  
2,3 Dpg

Abstract The addition of oxalate to blood stored in Citrate-phosphate-dextrose (CPD) produces a marked improvement in 2,3-diphosphoglycerate (2,3-DPG) preservation; an increase in 2,3-DPG levels can also be documented in short-term incubation studies. Oxalate is a potent in vitro inhibitor of red cell lactate dehydrogenase, monophosphoglycerate mutase, and pyruvate kinase (PK). In the presence of fructose 1,6-diphosphate the latter inhibitory effect is competitive with phospho(enol)pyruvate (PEP). Determination of the levels of intermediate compounds in red cells incubated with oxalate suggest the presence of inhibition at the PK step, indicating that this is the site of oxalate action. Apparent inhibition at the glyceraldehyde phosphate dehydrogenase step is apparently due to an increase in the NADH/NAD ratio. Oxalate had no effect on the in vivo viability of rabbit red cells stored in CPD preservatives for 21 days. Greater understanding of the toxicity of oxalate is required before it can be considered suitable as a component of preservative media, but appreciation of the mechanism by which it affects 2,3-DPG levels may be important in design of other blood additives. Malonate, the 3-carbon dicarboxylic acid analogue of oxalate late did not inhibit pyruvate kinase nor affect 2,3-DPG levels.

Postnatal regulation of canine oxygen delivery: control of erythrocyte 2,3-DPG levels

1982 ◽  
Vol 242 (4) ◽  
pp. H500-H506
Author(s):  
P. A. Mueggler ◽  
J. A. Black
Keyword(s):  
Pyruvate Kinase ◽  
Oxygen Affinity ◽  
Kinetic Properties ◽  
Intracellular Activity ◽  
Slow Decline ◽  
Blood Oxygen Affinity ◽  
Intracellular Concentrations ◽  
Glycolytic Rate ◽  
2,3 Dpg

The oxygen affinity of canine blood changes markedly following birth. These changes are correlated with alterations in the intracellular concentration of 2,3-diphosphoglycerate (2,3-DPG). We have examined the control of erythrocyte glycolysis by measurements of intracellular enzymes and intermediates, and we have identified the component responsible for regulation of 2,3-DPG concentration and hence blood oxygen affinity during canine postnatal development. The concentration of 2,3-DPG could be regulated entirely by the enzymes of the Rapoport-Luebering shunt. We have not detected any alterations in the levels or intracellular activity of 2,3-DPG mutase or 2,3-DPG phosphatase during development; therefore postnatal changes of 2,3-DPG must be a result of changes in the intracellular concentrations of 1,3-diphosphoglycerate (1,3-DPG) that are controlled by other reactions in the glycolytic pathway. Neither low intracellular concentrations of glucose, the glycolytic substrate, nor an inherently low glycolytic rate can account for the low 2,3-DPG levels at birth. 1,3-DPG concentrations and hence 2,3-DPG concentrations are controlled by the activity of pyruvate kinase, which acts as a glycolytic sink reaction. The intracellular activity of pyruvate kinase decreases during the first 50-60 days of age and causes the accumulation of 2,3-DPG. There is a subsequent change in the in vivo kinetic properties of the enzyme, giving increased intracellular activity and resulting in the slow decline of 2,3-DPG concentrations toward normal adult values.

scholarly journals Near-normal circulatory survival of rabbit red cells exposed to high levels of Ca and ionophore in vitro

Blood ◽  
1985 ◽  
Vol 66 (1) ◽  
pp. 220-223 ◽  
Author(s):  
RM Bookchin ◽  
EF Jr Roth ◽  
VL Lew
Keyword(s):  
Adenosine Triphosphate ◽  
Indirect Evidence ◽  
Red Cells ◽  
Ionophore A23187 ◽  
Red Cell ◽  
L Cells

Abstract The belief is widely held, on the basis of indirect evidence, that a substantial, even brief elevation of red cell Ca content must result in a marked shortening of circulatory survival. To test this notion directly, we exposed rabbit red cells in vitro to the ionophore A23187 and Ca so as to produce sustained uniform cell Ca levels of 40 to 360 mumol/L cells for one to 60 minutes, and compared the survival of the Ca-loaded cells in vivo with that of ionophore-treated controls, simultaneously, in the same rabbits. Despite marked reductions in cell adenosine triphosphate and dehydration of the Ca-exposed cells prior to reinfusion, the majority of cells, all of which had experienced these high cytoplasmic Ca levels, showed normal or near-normal survival in the circulation.

scholarly journals Pattern of pyruvate kinase isozymes in erythroleukemia cell lines and in normal human erythroblasts

Blood ◽  
1984 ◽  
Vol 64 (4) ◽  
pp. 930-936 ◽  
Author(s):  
I Max-Audit ◽  
U Testa ◽  
D Kechemir ◽  
M Titeux ◽  
W Vainchenker ◽  
...  
Keyword(s):  
Pyruvate Kinase ◽  
Cell Lines ◽  
Fetal Liver ◽  
Erythroid Cells ◽  
Low Concentrations ◽  
Erythroleukemia Cell ◽  
Erythroleukemic Cell ◽  
High Level

Abstract To further investigate the erythroid nature of the two human erythroleukemia cell lines, K562 and HEL-60, and to define the ontogeny of pyruvate kinase (PK) isozymes (R, M2) in developing human erythroid cells, we have studied the isozymic alterations, if any, during differentiation of these cell lines in vitro and normoblasts isolated from fetal liver in vivo. PK activity of erythroleukemic cell lines was intermediate between that observed in leukocytes and in fetal liver erythroblasts. These cell lines contained a high level of M2-PK, but R- PK was always present, albeit at low concentrations, in all the clones or subclones we studied. Erythroblasts from fetal liver were separated according to density on a Stractan gradient. R-PK levels were nearly constant in the different fractions, whereas M2-PK levels markedly decreased as the erythroblasts became mature and almost completely disappeared in late erythroid cells. Thus, these results clearly demonstrate the erythroid origin of these cell lines.

scholarly journals Tulathromycin Exerts Proresolving Effects in Bovine Neutrophils by Inhibiting Phospholipases and Altering Leukotriene B4, Prostaglandin E2, and Lipoxin A4Production

2014 ◽  
Vol 58 (8) ◽  
pp. 4298-4307 ◽  
Author(s):  
Carrie D. Fischer ◽  
Stephanie C. Duquette ◽  
Bernard S. Renaux ◽  
Troy D. Feener ◽  
Douglas W. Morck ◽  
...  
Keyword(s):  
Calcium Ionophore ◽  
Leukotriene B4 ◽  
Calcium Ionophore A23187 ◽  
Lipid Signaling ◽  
Prostaglandin E ◽  
Ionophore A23187 ◽  
Proinflammatory Mediators ◽  
Bovine Neutrophils

ABSTRACTThe accumulation of neutrophils and proinflammatory mediators, such as leukotriene B4(LTB4), is a classic marker of inflammatory disease. The clearance of apoptotic neutrophils, inhibition of proinflammatory signaling, and production of proresolving lipids (including lipoxins, such as lipoxin A4[LXA4]) are imperative for resolving inflammation. Tulathromycin (TUL), a macrolide used to treat bovine respiratory disease, confers immunomodulatory benefits via mechanisms that remain unclear. We recently reported the anti-inflammatory properties of TUL in bovine phagocytesin vitroand inMannheimia haemolytica-challenged calves. The findings demonstrated that this system offers a powerful model for investigating novel mechanisms of pharmacological immunomodulation. In the present study, we examined the effects of TUL in a nonbacterial model of pulmonary inflammationin vivoand characterized its effects on lipid signaling. In bronchoalveolar lavage (BAL) fluid samples from calves challenged with zymosan particles (50 mg), treatment with TUL (2.5 mg/kg of body weight) significantly reduced pulmonary levels of LTB4and prostaglandin E2(PGE2). In calcium ionophore (A23187)-stimulated bovine neutrophils, TUL inhibited phospholipase D (PLD), cytosolic phospholipase A2(PLA2) activity, and the release of LTB4. In contrast, TUL promoted the secretion of LXA4in resting and A23187-stimulated neutrophils, while levels of its precursor, 15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE], were significantly lower. These findings indicate that TUL directly modulates lipid signaling by inhibiting the production of proinflammatory eicosanoids and promoting the production of proresolving lipoxins.

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