scholarly journals Activation of protein kinase C in human neutrophils attenuates agonist-stimulated rises in cytosolic free Ca2+ concentration by inhibiting bivalent-cation influx and intracellular Ca2+ release in addition to stimulating Ca2+ efflux

1989 ◽  
Vol 264 (2) ◽  
pp. 357-364 ◽  
Author(s):  
S A McCarthy ◽  
T J Hallam ◽  
J E Merritt
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Kinase Inhibitor ◽  
Human Neutrophils ◽  
Bivalent Cation ◽  
Kinase Activation ◽  
Kinase C ◽  
Bivalent Cations ◽  
Intracellular Release ◽  
Stimulation Of

Stimulation of fura-2-loaded human neutrophils with formylmethionyl-leucyl-phenylalanine (FMLP) or ionomycin elevated the cytosolic free Ca2+ concentration, [Ca2+], to a maintained elevated level. Activation of protein kinase C (C-kinase) with phorbol 12-myristate 13-acetate, 4 beta-phorbol 12,13-didecanoate or dioctanoylglycerol caused decreases in [Ca2+]i from this level. 4 alpha-Phorbol didecanoate, which does not activate C-kinase, had no effect. These results confirm previous reports that C-kinase activation decreases neutrophil [Ca2+]i by stimulating removal of Ca2+ from the cytosol. Further experiments showed that activation of C-kinase attenuated the component of the FMLP-stimulated [Ca2+]i rise that was dependent on external Ca2+. C-kinase activation also inhibited FMLP-stimulated entry of the quenching cation, Mn2+, used as an indicator of bivalent-cation entry. In contrast, C-kinase activation caused only a partial inhibition of FMLP-stimulated release of Ca2+ from intracellular stores. 4 alpha-Phorbol didecanoate was ineffective in inhibiting Ca2+ entry, Mn2+ entry and intracellular Ca2+ release. Addition of FMLP also stimulated a decrease in the ionomycin-elevated [Ca2+]i, and this effect was blocked by staurosporine, a protein kinase inhibitor. These results show that, in addition to stimulating Ca2+ efflux, C-kinase activation in neutrophils inhibits FMLP-stimulated entry of bivalent cations, and partially inhibits intracellular release of Ca2+. Further, FMLP itself can modulate [Ca2+]i by activation of C-kinase.

The protein kinase C inhibitor H-7 activates human neutrophils: effect on shape, actin polymerization, fluid pinocytosis and locomotion

1990 ◽  
Vol 96 (1) ◽  
pp. 99-106
Author(s):  
H.U. Keller ◽  
V. Niggli ◽  
A. Zimmermann ◽  
R. Portmann
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Actin Polymerization ◽  
Kinase Inhibitor ◽  
Human Neutrophils ◽  
Kinase C ◽  
Chemotactic Peptides ◽  
Protein Kinase C Inhibitor ◽  
Shape Changes ◽  
Stimulation Of

The present study demonstrates new properties of H-7. The protein kinase inhibitor H-7 is a potent activator of several neutrophil functions. Stimulation of initially spherical nonmotile neutrophils elicits vigorous shape changes within a few seconds, increases in cytoskeletal actin, altered F-actin distribution, increased adhesiveness and a relatively small increase in pinocytic activity. H-7 has also chemokinetic activities. Depending on the experimental condition, H-7 may elicit or inhibit neutrophil locomotion. It failed to induce chemotaxis. Thus, the response pattern elicited by H-7 is different from that of other leukocyte activators such as chemotactic peptides, PMA or diacylglycerols. The finding that H-7 can elicit shape changes, actin polymerization and pinocytosis suggests that these events can occur without activation of protein kinase C (PKC). PMA-induced shape changes and stimulation of pinocytosis were not inhibited by H-7.

Role of protein kinase C in T-cell antigen receptor regulation of p21ras: evidence that two p21ras regulatory pathways coexist in T cells

1992 ◽  
Vol 12 (7) ◽  
pp. 3305-3312
Author(s):  
M Izquierdo ◽  
J Downward ◽  
J D Graves ◽  
D A Cantrell
Keyword(s):  
T Cells ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Kinase Inhibitor ◽  
Antigen Receptor ◽  
Permeabilized Cell ◽  
Regulatory Pathways ◽  
Kinase C ◽  
Stimulation Of

T-lymphocyte activation via the antigen receptor complex (TCR) results in accumulation of p21ras in the active GTP-bound state. Stimulation of protein kinase C (PKC) can also activate p21ras, and it has been proposed that the TCR effect on p21ras occurs as a consequence of TCR regulation of PKC. To test the role of PKC in TCR regulation of p21ras, a permeabilized cell system was used to examine TCR regulation of p21ras under conditions in which TCR activation of PKC was blocked, first by using a PKC pseudosubstrate peptide inhibitor and second by using ionic conditions that prevent phosphatidyl inositol hydrolysis and hence diacylglycerol production and PKC stimulation. The data show that TCR-induced p21ras activation is not mediated exclusively by PKC. Thus, in the absence of PKC stimulation, the TCR was still able to induce accumulation of p21ras-GTP complexes, and this stimulation correlated with an inactivation of p21ras GTPase-activating proteins. The protein tyrosine kinase inhibitor herbimycin could prevent the non-PKC-mediated, TCR-induced stimulation of p21ras. These data indicate that two mechanisms for p21ras regulation coexist in T cells: one PKC mediated and one not. The TCR can apparently couple to p21ras via a non-PKC-controlled route that may involve tyrosine kinases.

d-Glucose and insulin stimulate migration and tubular formation of human endothelial cells in vitro

1999 ◽  
Vol 277 (3) ◽  
pp. E433-E438 ◽  
Author(s):  
Satoshi Shigematsu ◽  
Keishi Yamauchi ◽  
Kohji Nakajima ◽  
Sachiko Iijima ◽  
Toru Aizawa ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Cell Migration ◽  
Endothelial Cell ◽  
Protein Kinase ◽  
Kinase Inhibitor ◽  
Endothelial Cell Migration ◽  
Insulin Effect ◽  
Kinase C ◽  
Tubular Formation ◽  
Stimulation Of

Effects of highd-glucose and insulin on the endothelial cell migration and tubular formation were investigated with the use of ECV304 cells, a clonal human umbilical cord endothelial cell line. Exposure of the cells to highd-glucose resulted in a marked increase in the migration, which was blocked by inhibitors of protein kinase C such as H7 (10 μM) and GF109203X (200 nM). Furthermore, a protein kinase C agonist, phorbol 12-myristate 13-acetate, had an effect similar to that of glucose on ECV304 cells. Glucose stimulation of the migration was additively enhanced by 100 nM insulin, and the insulin effect was found to be unaffected by either PD-98059 or wortmannin, a mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase inhibitor and a phosphatidylinositol 3-kinase inhibitor, respectively. Neither did H7 inhibit insulin stimulation of the migration. In contrast, a combination of high d-glucose and insulin, rather than either one alone, promoted tubular formation, which was inhibited by addition of 10 μM PD-98059. Stimulation of ECV304 cells by the combination of highd-glucose and insulin also caused an activation of MAPK, which was again obliterated by the same concentration of PD-98059. In conclusion, human endothelial cell migration and tubular formation are stimulated by highd-glucose and insulin in different ways. In the former reaction, either is effective, a combination of the two results in an additive effect, and activation of protein kinase C is involved. In contrast, tubular formation will only occur in the presence of a combination of highd-glucose and insulin, and MAPK plays an essential role.

scholarly journals Epidermal growth factor stimulation of stromelysin mRNA in rat fibroblasts requires induction of proto-oncogenes c-fos and c-jun and activation of protein kinase C.

1990 ◽  
Vol 10 (8) ◽  
pp. 4284-4293 ◽  
Author(s):  
S E McDonnell ◽  
L D Kerr ◽  
L M Matrisian
Keyword(s):  
Protein Kinase C ◽  
Growth Factor ◽  
Protein Kinase ◽  
Epidermal Growth Factor ◽  
Kinase Activation ◽  
Kinase C ◽  
Protein Kinase C Activity ◽  
Epidermal Growth ◽  
Rat Fibroblasts ◽  
Stimulation Of

Stromelysin (transin) is a secreted metalloprotease that is transcriptionally induced by a variety of growth factors and oncogenes. We examined the necessity of specific secondary (protein kinase C) and tertiary (c-fos and c-jun protein products) messengers in the transactivation of stromelysin gene expression by epidermal growth factor (EGF). Rat-1 fibroblasts exposed to antisense c-fos DNA or RNA demonstrated that c-fos expression was necessary for complete EGF induction of stromelysin expression. Similar results demonstrating the necessity of c-jun protein in the EGF induction of stromelysin were obtained. We also demonstrated that protein kinase C activation is required for the EGF induction of stromelysin, since phorbol ester desensitization of C kinase proteins abolished the ability of EGF to induce stromelysin mRNA, protein, and promoter activity. In reconstitution experiments, neither c-fos, c-jun, nor C kinase activation alone induced significant stromelysin expression. Overexpression of c-fos and c-jun was able to induce stromelysin to a level similar to that of the growth factor, and stimulation of protein kinase C activity augmented this induction. The data suggest that the EGF induction of stromelysin in rat fibroblasts procedes through a pathway involving c-fos, c-jun, and protein kinase C.

scholarly journals Involvement of calcium in modulation of neutrophil function by phorbol esters that activate protein kinase C isotypes and related enzymes

1993 ◽  
Vol 289 (3) ◽  
pp. 919-926 ◽  
Author(s):  
J E Merritt ◽  
K E Moores ◽  
A T Evans ◽  
P Sharma ◽  
F J Evans ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phorbol Esters ◽  
Biological Activities ◽  
Inhibitory Effect ◽  
Human Neutrophils ◽  
Published Data ◽  
Bivalent Cation ◽  
Kinase C

In this study, the effects of a series of phorbol esters with different spectra of biological activities and different patterns of activation of the isoenzymes of protein kinase C (PKC) have been studied in human neutrophils. The aim was to gain more information on which isoenzymes of PKC are involved in neutrophil activation, specifically inhibition of fMet-Leu-Phe (fMLP)-stimulated bivalent cation influx and stimulation of O2-. release (either alone or potentiation of the response to fMLP). Prior addition of both phorbol 12-myristate 13-acetate (PMA) and sapintoxin A (SAPA) inhibited fMLP-stimulated Mn2+ influx. Higher concentrations of resiniferatoxin (RX) were also inhibitory, inhibition being more apparent at longer preincubation times. However, 12-deoxyphorbol 13-O-phenylacetate (DOPPA) showed only a slight inhibitory effect and required a prolonged preincubation. PMA, SAPA and RX, but not DOPPA, stimulated O2-. release by themselves. Lower concentrations of PMA, SAPA and RX, which were ineffective alone, considerably potentiated O2-. release stimulated by fMLP, whereas DOPPA had little or no effect. These results rule out a major role for PKC-delta (not activated by SAPA) and PKC-beta 1 (activated by DOPPA), but suggest the involvement of RX kinase in addition to PKC in the inhibition of fMLP-stimulated Mn2+ influx and potentiation of fMLP-stimulated O2-. release. However, when the cytosolic free Ca2+ concentration ([Ca2+]i) was elevated with the Ca2+ ionophore ionomycin, DOPPA was able to stimulate O2-. release, which probably reflects the known Ca2+ requirement for activation of PKC-beta 1 by DOPPA in vitro. The effects of the other phorbols were also enhanced when [Ca2+]i was elevated; all of the phorbols synergize, to variable extents, with Ca2+ to activate PKC in vitro. Enhancement of RX-stimulated O2- release by elevation of [Ca2+]i was unexpected, since RX kinase has been reported to be inhibited by high concentrations of Ca2+ in vitro. Finally, use of fura-2 and SK&F 96365 to manipulate the fMLP-stimulated rise in [Ca2+]i showed that when fMLP was able to evoke its normal rise in [Ca2+]i (to a peak of 700-900 nM), O2-. release was potentiated by PMA, SAPA and RX. However, when fMLP was only able to evoke a small increase in [Ca2+]i (to a peak of 400 nM), potentiation by PMA was unaffected but potentiation by SAPA and RX was considerably reduced. This observation agrees with published data demonstrating that activation of PKC in vitro by SAPA is more Ca(2+)-dependent than activation by PMA.(ABSTRACT TRUNCATED AT 400 WORDS)

Bradykinin-stimulated arachidonic acid release from MDCK cells is not protein kinase C dependent

1997 ◽  
Vol 273 (5) ◽  
pp. C1605-C1612 ◽  
Author(s):  
Chris R. J. Kennedy ◽  
Richard L. Hébert ◽  
Minh T. Do ◽  
Pierre R. Proulx
Keyword(s):  
Arachidonic Acid ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Kinase Inhibitor ◽  
Active Form ◽  
Mitogen Activated Protein Kinase ◽  
Cell Extracts ◽  
Kinase C ◽  
A 23187 ◽  
Stimulation Of

Bradykinin (BK)-induced release of arachidonic acid (AA) from Madin-Darby canine kidney (MDCK) D1 cells was investigated. Phorbol 12-myristate 13-acetate (PMA) caused a synergistic increase in BK- and A-23187-induced release of AA but alone had no effect on this release. Inhibition of protein kinase C (PKC) with bisindolmaleimide I (BIS) abolished the synergistic effects of PMA but did not affect AA release caused by BK or A-23187 alone. Downregulation of PKC with 100 nM PMA resulted in a reduction of AA release induced by BK or A-23187 addition, which corresponded to a decrease in cytoplasmic phospholipase A2(cPLA2) activity as measured in cell extracts. Although Western blotting revealed no differences in cPLA2 expression as a result of PMA treatment, phosphorylation of the enzyme, as assessed by phosphoserine content, was significantly reduced in PKC-depleted cells. These results imply that, with PKC downregulation, subsequent BK stimulation results in a Ca2+-dependent translocation of a less phosphorylated, less active form of cPLA2. Any stimulation of PKC by BK addition did not appear as a significant event in onset reponses leading to AA release. On the other hand, inhibition of the mitogen-activated protein kinase (MAPK) cascade with the MAPK kinase inhibitor, PD-98059, significantly decreased BK-induced release of AA, a finding that, with our other results, points to the existence of a PKC-independent route for stimulation of MAPK and the propagation of onset responses.

scholarly journals Effects of chemotactic factors and other agents on the amounts of actin and a 65,000-mol-wt protein associated with the cytoskeleton of rabbit and human neutrophils.

1985 ◽  
Vol 101 (1) ◽  
pp. 182-188 ◽  
Author(s):  
R Yassin ◽  
J Shefcyk ◽  
J R White ◽  
W Tao ◽  
M Volpi ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Leukotriene B4 ◽  
Intracellular Concentration ◽  
Human Neutrophils ◽  
Free Calcium ◽  
High Osmolarity ◽  
Kinase C ◽  
Chemotactic Factors ◽  
Stimulation Of

Stimulation of rabbit neutrophils by the chemotactic factors fMet-Leu-Phe and leukotriene B4, by platelet activating factor, or by arachidonic acid produces a rapid and dose-dependent increase in the amounts of actin and of a 65,000-mol-wt protein associated with the cytoskeleton. Phorbol 12-myristate, 13-acetate, the calcium ionophore A23187 in the presence or absence of EGTA, and the fluorescent calcium chelator quin-2 also cause an increase in cytoskeletal actin. The stimulated increases in the cytoskeletal actin are not dependent on a rise in the intracellular concentration of free calcium and are not mediated by an increase in the intracellular pH or activation of protein kinase C. The increases in the cytoskeletal actin produced by fMet-Leu-Phe and leukotriene B4, but not by phorbol 12-myristate, 13-acetate, are inhibited by high osmolarity. The effect of hyperosmolarity requires a decrease in cell volume, is not mediated by an increase in basal intracellular concentration of free calcium, and is not prevented by pretreating the cells with amiloride. Preincubation of the cells with hyperosmotic solution also inhibits degranulation produced by all the stimuli tested. The inhibitory action of high osmolarity on the fMet-Leu-Phe and leukotriene B4 induced stimulation of cytoskeletal actin is discussed in terms of the possibility that the addition of high osmolarity, either directly or through activation of protein kinase C, causes receptor uncoupling.

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