scholarly journals Induction of the differentiation of HL-60 cells by phorbol 12-myristate 13-acetate activates a Na+-dependent uridine-transport system. Involvement of protein kinase C

1991 ◽  
Vol 274 (1) ◽  
pp. 85-90 ◽  
Author(s):  
C W Lee ◽  
J A Sokoloski ◽  
A C Sartorelli ◽  
R E Handschumacher
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Nucleoside Transport ◽  
Facilitated Diffusion ◽  
Similar Increase ◽  
Monocytic Differentiation ◽  
Cell Water ◽  
Cellular Mechanisms ◽  
Kinase C ◽  
Dependent Transport

The Na(+)-dependent transport and facilitated diffusion of uridine were measured after differentiation of HL-60 leukaemia cells along the monocytic pathway by phorbol 12-myristate 13-acetate (PMA). PMA (200 ng/ml) caused a marked increase in Na(+)-dependent uridine transport within 48 h of exposure that was attributable to an increase in transport affinity (apparent Km values of 1.15 +/- 0.22 and 44 +/- 4.4 microM for PMA-induced and uninduced cells respectively), with no change in Vmax. (0.15 +/- 0.02 and 0.13 +/- 0.01 pmol/s per microliter of cell water for PMA-induced and uninduced cells respectively). A corresponding rapid decrease in both the rate of facilitated diffusion and the formation of uracil nucleotides occurred in PMA-induced cells. As a consequence of these changes, intracellular pools of uridine 3-4-fold greater than those in the medium were generated. A similar increase in Na(+)-dependent transport of adenosine, inosine, guanosine, thymidine and cytidine (Km values of 1-4 microM) was observed. The effects of PMA on the activation of the Na(+)-dependent uridine transporter were inhibited by staurosporine, suggesting the involvement of protein kinase C. The findings indicate that a change in the balance of the cellular mechanisms employed for nucleoside transport occurs during the monocytic differentiation of HL-60 leukaemia cells.

scholarly journals Decrease in equilibrative uridine transport during monocytic differentiation of HL-60 leukaemia: involvement of protein kinase C

1994 ◽  
Vol 300 (2) ◽  
pp. 407-412 ◽  
Author(s):  
C W Lee
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Binding Sites ◽  
Plasma Membranes ◽  
Nucleoside Transport ◽  
Protective Effects ◽  
Transport Activity ◽  
Monocytic Differentiation ◽  
Response Curves ◽  
Kinase C

The dose-response curves for the inhibition of equilibrative uridine transport by dilazep, dipyridamole and nitrobenzylthioinosine (NBMPR) in undifferentiated HL-60 cells were biphasic. Some 70% of the transport activity was inhibited with IC50 values of 0.7, 1 and 7 nM respectively. No inhibition of the remaining 30% of transport activity was observed until the dilazep, dipyridamole and NBMPR concentrations exceeded 1, 0.1 and 3 microM respectively. Exposure to phorbol 12-myristate 13-acetate (PMA) for 48 h, to induce monocytic differentiation, caused a 20-fold decrease in Vmax. of both NBMPR-sensitive and NBMPR-insensitive equilibrative uridine transport. The decrease in NBMPR-sensitive uridine transport induced by PMA corresponded to a decrease in NBMPR binding sites. A 30% decrease in specific NBMPR binding sites occurred within 6 h of PMA exposure, and could be prevented by uridine and thymidine at concentrations as low as 100 microM, and by staurosporine at 40 nM. However, the protective effects of these compounds diminished with prolonged PMA exposure. No protection was observed with uracil. Exogenous protein kinase C (PKC) in the presence of ATP and PMA decreased the number of specific NBMPR-binding sites in purified HL-60 cell plasma membranes. These results suggest that a PKC-induced conformational change in substrate-binding/transporting site may be responsible for the decrease in NBMPR-sensitive nucleoside transport during PMA-induced monocytic differentiation of HL-60 cells.

Mechanism of negative lusitropic effect of alpha 1-adrenoceptor stimulation in cat papillary muscles

1996 ◽  
Vol 270 (2) ◽  
pp. H701-H709 ◽  
Author(s):  
M. Vila-Petroff ◽  
G. N. Perez ◽  
B. Alvarez ◽  
H. E. Cingolani ◽  
A. Mattiazzi
Keyword(s):  
Protein Kinase C ◽  
Relaxation Time ◽  
Protein Kinase ◽  
Maximal Velocity ◽  
Papillary Muscles ◽  
Similar Increase ◽  
Kinase C ◽  
Maximal Tension ◽  
Pkc Inhibitors ◽  
Pkc Activation

Experiments were performed in cat papillary muscles to explore the mechanisms by which alpha 1-adrenoceptor stimulation affects myocardial relaxation. Phenylephrine (PE; 10 microM) + atenolol (1 microM; n = 8 experiments) produced a negative lusitropic effect, i.e., a prolongation of half-relaxation time (t1/2; time to 50% relaxation) by 30 +/- 10% (P < 0.05) and a proportionally smaller increase in maximal velocity of relaxation (-T) than in maximal velocity of contraction (+T), which significantly increased the ratio +T/-T. A similar increase in contractility, produced by increasing calcium, failed to significantly change t1/2 and +T/-T. PE-induced negative lusitropic effect was significantly inhibited by two protein kinase C (PKC) inhibitors, staurosporine (0.1 microM) and chelerythrine (10 microM). PE also increased intracellular pH by 0.18 +/- 0.05 pH units (P < 0.05, n = 4), as measured by the fluorescent dye 2'-7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Intracellular alkalosis and the negative lusitropic effect of PE were prevented by the Na+/H+ exchanger inhibitor ethylisopropylamiloride (10 microM). No significant changes in calcium myofilament sensitivity and maximal tension were detected in trabeculae treated with PE either before or after chemical skinning. These results indicate that a Na+/H+ exchanger-induced intracellular alkalosis, possibly mediated by PKC activation, may fully account for the negative lusitropism of alpha 1-adrenoceptor stimulation.

Reactive oxygen-mediated contraction in pulmonary arterial smooth muscle: cellular mechanisms

1991 ◽  
Vol 69 (3) ◽  
pp. 383-388 ◽  
Author(s):  
N. Jin ◽  
C. S. Packer ◽  
R. A. Rhoades
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Reactive Oxygen ◽  
Oxygen Species ◽  
Cellular Mechanisms ◽  
Kinase C ◽  
Pulmonary Arterial ◽  
Pulmonary Arterial Smooth Muscle ◽  
Oxidase Reaction

Reactive oxygen species (at least relatively high doses) cause contraction of pulmonary arterial smooth muscle. The objective of the present study was to elucidate the possible cellular mechanisms involved in reactive oxygen-mediated contraction. Isolated arterial rings from Sprague–Dawley rats were placed in tissue baths containing Earle's balanced salt solution. The maximum active force production (Po) in response to 80 mM KCl was obtained. All other responses were normalized as percentages of Po for comparative purposes. Exposure to reactive oxygen (generated from either the xanthine oxidase reaction (XO) or the glucose oxidase reaction) resulted in pulmonary arterial muscle developing mean active tension of 17.1 ± 3.0% Po. This contraction was independent of extracellular calcium, since it was not affected by verapamil (a calcium channel blocker) or by placement of the arterial muscle in calcium-free media. Phentolamine (an α1-receptor blocker) and propranolol (a β-receptor blocker) did not diminish the response to XO. Ryanodine (a SR calcium release inhibitor), while reducing the response to norepinephrine, did not affect the response to XO. However, H-7 (an inhibitor of protein kinase C) decreased the XO-mediated contraction by 49%. These results indicate that while Ca2+ may not be involved as a second messenger, protein kinase C activity appears to play a role in the transduction pathway of reactive oxygen species mediated contraction of pulmonary arterial smooth muscle.Key words: muscle calcium, α1-receptor, ryanodine, protein kinase C, vascular smooth muscle, oxygen radicals, verapamil.

Transport characteristics of HL-1 cells: a new model for the study of adenosine physiology in cardiomyocytes

2002 ◽  
Vol 80 (5) ◽  
pp. 655-665 ◽  
Author(s):  
Naz Chaudary ◽  
Irina Shuralyova ◽  
Tamar Liron ◽  
Gary Sweeney ◽  
Imogen R Coe
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Glucose Transport ◽  
Cell Model ◽  
Polymerase Chain Reaction Analysis ◽  
Receptor Activation ◽  
Nucleoside Transport ◽  
Metabolic Enzyme ◽  
Mouse Heart ◽  
Kinase C

Adenosine is a physiologically important nucleoside in the cardiovascular system where it can act as a cardioprotectant and modulator of energy usage. Adenosine transporters (ATs) modulate cellular adenosine levels, which, in turn, can affect a number of processes such as receptor activation and glucose uptake, but their role in cardiac physiology is poorly understood. Therefore, we have developed a new cell model by determining various adenosine-related characteristics of HL-1, an immortalized atrial cardiomyocyte murine cell line. Adenosine uptake in HL-1 cells is sodium independent, saturable, and inhibitable by nucleoside transport inhibitors (nitrobenzylthioinosine (NBTI), dipyridamole, dilazep). Reverse transcription – polymerase chain reaction analysis confirmed that HL-1 cells possess mouse equilibrative nucleoside transporters 1 and 2 (mENT1, mENT2) and kinetic analyses indicate moderate-affinity (Km = 51.3 ± 12.9 μM), NBTI-sensitive adenosine transport. NBTI binds at a high-affinity single site (Bmax = 520 ± 10 fmol/mg protein, Kd = 0.11 ± 0.04 nM, 1.6 × 105 NBTI-binding sites/cell). HL-1 cells possess adenosine receptor, metabolic enzyme, protein kinase C isoform, and insulin-stimulated glucose transport profiles that match normal mouse heart. Therefore, HL-1 is an excellent model to study ATs within cardiomyocytes and the first model for evaluating in detail the role of the ATs in modulating effects of adenosine.Key words: adenosine, nucleoside transport, HL-1 cells, cardiovascular, glucose transport, protein kinase C.

scholarly journals Bryostatin 1 activates protein kinase C and induces monocytic differentiation of HL-60 cells

Blood ◽  
1988 ◽  
Vol 72 (1) ◽  
pp. 208-213 ◽  
Author(s):  
RM Stone ◽  
E Sariban ◽  
GR Pettit ◽  
DW Kufe
Keyword(s):  
Gene Expression ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Growth Inhibition ◽  
Phorbol Ester ◽  
Phorbol Esters ◽  
Cell Protein ◽  
Monocytic Differentiation ◽  
Bryostatin 1 ◽  
Kinase C

Phorbol esters induce the human HL-60 promyelocytic cell line to differentiate along a monocytic pathway. This induction of differentiation may involve phorbol ester-induced activation of the phospholipid- and calcium-dependent protein kinase C. Bryostatin 1, a macrocyclic lactone, has been shown to compete with phorbol esters for binding to protein kinase C. We have confirmed that bryostatin 1 translocates activity of protein kinase C from the cytosolic to membrane fractions of HL-60 cells. The present results also demonstrate that bryostatin 1 (10 nmol/L) induces monocytic differentiation of HL- 60 cells as determined by adherence, growth inhibition, appearance of monocyte cell surface antigens, and alpha-naphthyl acetate esterase staining. Furthermore, bryostatin 1 (10 nmol/L) downregulated c-myc expression and induced c-fos, c-fms, and tumor necrosis factor transcripts. These changes in gene expression induced by bryostatin 1 are similar to those associated with phorbol ester-induced monocytic differentiation of HL-60 cells. In contrast, exposure to a higher concentration of bryostatin 1 (100 nmol/L) had less of an effect on growth inhibition of HL-60 cells and changes in gene expression. Moreover, 100 nmol/L bryostatin 1 antagonized the cytostatic effects and adherence induced by phorbol esters. Our results thus suggest that bryostatin 1 activates HL-60 cell protein kinase C and that this effect is associated with induction of monocytic differentiation.

scholarly journals Phospholipase C activates protein kinase C and induces monocytic differentiation of HL-60 cells

Blood ◽  
1988 ◽  
Vol 72 (2) ◽  
pp. 739-744 ◽  
Author(s):  
RM Stone ◽  
BL Weber ◽  
DR Spriggs ◽  
DW Kufe
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Inositol Phosphates ◽  
Cell Protein ◽  
Mrna Levels ◽  
Monocytic Differentiation ◽  
Cell Surface Antigens ◽  
Monocytic Cell ◽  
Kinase C

Phospholipase C (PLC)-mediated hydrolysis of membrane phospholipids results in the production of diacylglycerol, inositol phosphates, and choline metabolites. Inositol triphosphate increases calcium levels, while diacylglycerol activates protein kinase C. The present studies demonstrate that exogenous PLC generates inositol phosphates, releases choline metabolites, and activates protein kinase C in human HL-60 promyelocytic leukemia cells. PLC also induced monocytic differentiation of HL-60 cells as manifested by adherence, growth inhibition, and appearance of monocytic cell surface antigens. Furthermore, PLC treatment decreased c-myc mRNA levels and induced c- fos, c-fms, and tumor necrosis factor transcripts. The changes in gene expression induced by PLC are similar to those previously shown to be associated with phorbol ester-induced monocytic differentiation of HL- 60 cells. Our results thus demonstrate that exogenous PLC activates HL- 60 cell protein kinase C and that this effect is associated with induction of monocytic differentiation. PLC may therefore play a role in transducing signals from physiological inducers of monocytic differentiation.

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