Inhibition of calcium flux and calcium channel antagonist binding in the PC12 neural cell line by phorbol esters and protein kinase C

Phorbol esters are potent tumor promoters that have been widely used in studies of transmembrane signaling because of their ability to activate protein kinase C. To study the effect of phorbol esters (and indirectly, the role of protein kinase C) on cardiac muscle contractility, we examined the effects of phorbol myristate acetate (PMA) on contractile state, transmembrane 45Ca fluxes, and cytosolic free Ca concentration ([Ca]i) using spontaneously contracting cultured chick ventricular cells. PMA produced a concentration- and time-dependent decrease in the amplitude of cell motion [half maximum inhibitory concentration (IC50) = 130 nM] with maximal effect (54 +/- 5% of control) observed at 1 microM. PMA (1 microM) reduced 45Ca uptake rate by 16 +/- 4% (P less than 0.05) and the size of the rapidly exchangeable Ca pool by 11 +/- 2% (P less than 0.05) but did not alter the 45Ca efflux rate. In fura-2-loaded cells, PMA produced a decrease in [Ca]i from 96 +/- 7 to 72 +/- 5 nM (mean +/- SE; P less than 0.05) with a time course similar to that of alteration in contractile amplitude. PMA had no effect on cellular Na content. Phorbol didecanoate (1 microM), a phorbol diester that does not activate protein kinase C, produced no significant changes in contractile amplitude, 45Ca fluxes, or [Ca]i. These results indicate that PMA influences transsarcolemmal Ca uptake, and thus the excitation-contraction process, and suggest that protein kinase C may modulate myocardial Ca homeostasis and contractile state.

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Protein kinase C-dependent and -independent effects of phorbol esters on hippocampal calcium channel current

Journal of Neuroscience ◽

10.1523/jneurosci.10-05-01699.1990 ◽

1990 ◽

Vol 10 (5) ◽

pp. 1699-1706 ◽

Cited By ~ 46

Author(s):

D Doerner ◽

M Abdel-Latif ◽

TB Rogers ◽

BE Alger

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Calcium Channel ◽

Phorbol Esters ◽

Channel Current ◽

Kinase C ◽

Independent Effects

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Evidence that interleukin-1 and phorbol esters activate NF-kappa B by different pathways: role of protein kinase C.

Cell Regulation ◽

10.1091/mbc.2.4.329 ◽

1991 ◽

Vol 2 (4) ◽

pp. 329-335 ◽

Cited By ~ 46

Author(s):

K Bomsztyk ◽

J W Rooney ◽

T Iwasaki ◽

N A Rachie ◽

S K Dower ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Cell Line ◽

Phorbol Esters ◽

Interleukin 1 ◽

Interleukin 2 ◽

Nf Kappa B ◽

Kinase C ◽

Protein Kinase C Inhibitor

Nuclear factor kappa B (NF-kappa B) is a ubiquitous transcription factor that affects expression of many genes, including immunoglobulin kappa (kappa), the interleukin-2 receptor alpha chain, and two genes in HIV-1. NF-kappa B can be activated by a number of stimuli, including pharmacological stimulation of protein kinase C by phorbol 12-myristate 13-acetate (PMA) and treatment in vitro with either protein kinase C or protein kinase A. This has lead to the proposal that these kinases are key enzymes in the physiological activation of NF-kappa B as well. We have used a murine B cell line, 70Z/3, and T cell line, EL-4 6.1 C10, to study the activation of NF-kappa B by two physiological activators, interleukin-1 alpha (IL-1) and lipopolysaccharide (LPS). There are four reasons to propose that these agents activate pathways that do not include protein kinase C as a major component in these cell lines. First, the protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) strongly inhibited PMA-induced activation of NF-kappa B in 70Z/3 cells but had no effect on NF-kappa B activated by IL-1 or LPS. Second, depletion of protein kinase C by prolonged growth of 70Z/3 in PMA abrogated the capacity of the cells to activate NF-kappa B in response to further PMA treatment. However, these same cells activated NF-kappa B normally after either IL-1 or LPS treatment. Third, IL-1 effectively activated NF-kappa B in EL-4 6.1 C10 cells, but PMA did not. Fourth, interferon-gamma is a potent activator of protein kinase C in 70Z/3 cells, but is completely inactive in the mobilization of NF-kappa B. These results suggest that the physiological inducers IL-1 and LPS activate NF-kappa B by pathways independent of protein kinase C in both 70Z/3 and EL-4 6.1 C10 cells.

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Phorbol esters, but not the hormonal form of vitamin D, induce changes in protein kinase C during differentiation of human histiocytic lymphoma cell line (U-937)

Life Sciences ◽

10.1016/0024-3205(87)90105-6 ◽

1987 ◽

Vol 40 (21) ◽

pp. 2111-2117 ◽

Cited By ~ 5

Author(s):

G. Mezzetti ◽

G.P. Bagnara ◽

M.G. Monti ◽

L. Pernecco Casolo ◽

L. Bonsi ◽

...

Keyword(s):

Vitamin D ◽

Protein Kinase C ◽

Protein Kinase ◽

Cell Line ◽

Phorbol Esters ◽

Lymphoma Cell ◽

Lymphoma Cell Line ◽

Kinase C ◽

Histiocytic Lymphoma

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Differential effect of phorbol esters and interleukin-3 on protein kinase C isoform content and kinase activity in the FDC-P1 cell line

Blood ◽

10.1182/blood.v78.10.2633.bloodjournal78102633 ◽

1991 ◽

Vol 78 (10) ◽

pp. 2633-2641

Author(s):

DK Ways ◽

W Qin ◽

RS Riddle ◽

TD Garris ◽

TE Bennett ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Cell Line ◽

Kinase Activity ◽

Phorbol Esters ◽

Dependent Manner ◽

Interleukin 3 ◽

Kinase C ◽

Endogenous Substrates ◽

Cells Cultured

FD/PMA is a subclone of the interleukin-3 (IL-3)-dependent, FDC-P1 cell line, which proliferates in response to either 12-O- tetradecanoylphorbol-13 acetate (PMA) or IL-3. While several endogenous substrates were phosphorylated in response to protein kinase C (PKC) activation in FDC-P1, phospholipid-dependent phosphorylation in the FD/PMA grown in PMA was not observed. Basal, phosphatidylserine- independent, and diolein-independent phosphorylation of cytosolic substrates with molecular weights of 17, 52, 57, and 105 Kd were enhanced in FD/PMA cells grown in PMA as compared with FDC-P1 cells cultured in IL-3. Phosphorylation of a 105-Kd substrate was enhanced in the particulate fraction of FD/PMA cells maintained in PMA. The 17-Kd substrate in FD/PMA cells comigrated with a substrate phosphorylated in a PKC-dependent manner in FDC-P1 cells. Phosphorylation of the 52- and 57-Kd substrates, but not of the 17-Kd substrate, was inhibited by H-7 and staurosporine. A portion of the PMA-induced cytosolic kinase activity coeluted with PKC on diethyl aminoethyl chromatography. While FD/PMA cells cultured in PMA contained negligible PKC-dependent phosphorylation of endogenous substrates or histone, alpha and epsilon PKC isoforms were detected by Western blot analysis. PKC phosphotransferase activity was observed in FD/PMA cells grown in PMA when peptides corresponding to residues 720 to 737 of PKC-epsilon or residues 4 to 14 of myelin basic protein were used as substrates. These data indicate that maintenance of FD/PMA cells in PMA stimulates proliferation and markedly alters PKC substrate specificity. Generation of at least two phospholipid-independent kinases occurs in PMA-treated cells.

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Differential effect of phorbol esters and interleukin-3 on protein kinase C isoform content and kinase activity in the FDC-P1 cell line

Blood ◽

10.1182/blood.v78.10.2633.2633 ◽

1991 ◽

Vol 78 (10) ◽

pp. 2633-2641 ◽

Cited By ~ 8

Author(s):

DK Ways ◽

W Qin ◽

RS Riddle ◽

TD Garris ◽

TE Bennett ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Cell Line ◽

Kinase Activity ◽

Phorbol Esters ◽

Dependent Manner ◽

Interleukin 3 ◽

Kinase C ◽

Endogenous Substrates ◽

Cells Cultured

Abstract FD/PMA is a subclone of the interleukin-3 (IL-3)-dependent, FDC-P1 cell line, which proliferates in response to either 12-O- tetradecanoylphorbol-13 acetate (PMA) or IL-3. While several endogenous substrates were phosphorylated in response to protein kinase C (PKC) activation in FDC-P1, phospholipid-dependent phosphorylation in the FD/PMA grown in PMA was not observed. Basal, phosphatidylserine- independent, and diolein-independent phosphorylation of cytosolic substrates with molecular weights of 17, 52, 57, and 105 Kd were enhanced in FD/PMA cells grown in PMA as compared with FDC-P1 cells cultured in IL-3. Phosphorylation of a 105-Kd substrate was enhanced in the particulate fraction of FD/PMA cells maintained in PMA. The 17-Kd substrate in FD/PMA cells comigrated with a substrate phosphorylated in a PKC-dependent manner in FDC-P1 cells. Phosphorylation of the 52- and 57-Kd substrates, but not of the 17-Kd substrate, was inhibited by H-7 and staurosporine. A portion of the PMA-induced cytosolic kinase activity coeluted with PKC on diethyl aminoethyl chromatography. While FD/PMA cells cultured in PMA contained negligible PKC-dependent phosphorylation of endogenous substrates or histone, alpha and epsilon PKC isoforms were detected by Western blot analysis. PKC phosphotransferase activity was observed in FD/PMA cells grown in PMA when peptides corresponding to residues 720 to 737 of PKC-epsilon or residues 4 to 14 of myelin basic protein were used as substrates. These data indicate that maintenance of FD/PMA cells in PMA stimulates proliferation and markedly alters PKC substrate specificity. Generation of at least two phospholipid-independent kinases occurs in PMA-treated cells.

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Dual role for protein kinase C α as a regulator of ion secretion in the HT29cl.19A human colonic cell line

Biochemical Journal ◽

10.1042/bj2850673 ◽

1992 ◽

Vol 285 (2) ◽

pp. 673-679 ◽

Cited By ~ 19

Author(s):

N van den Berghe ◽

A B Vaandrager ◽

A G M Bot ◽

P J Parker ◽

H R de Jonge

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Cell Line ◽

Phorbol Esters ◽

Prolonged Exposure ◽

Electrical Response ◽

Short Circuit ◽

Ion Secretion ◽

Kinase C ◽

Pkc Alpha

The involvement of protein kinase C (PKC) in the regulation of intestinal ion secretion was studied in polarized monolayers of the HT29cl.19A human colon carcinoma cell line. Carbachol, phorbol esters [PMA (phorbol 12-myristate 13-acetate) and PDB (phorbol 12,13-dibutyrate)] and 8-bromo cyclic AMP (8-Br-cAMP) induced Cl secretion, as measured by a rise in the short-circuit current (ISC). The electrical response to carbachol coincided with a transient translocation of PKC alpha from the soluble to the particulate fraction. The carbachol-, PDB- and 8-Br-cAMP-induced ISC responses were inhibited by pretreatment of the cells with PMA (0.5 microM) for 2 h, a time period in which PKC alpha, beta 1 and gamma levels were not changed. As shown by 86Rb+ and 125I- efflux studies, the main targets for this inhibition were basolateral K+ transporters rather than apical Cl- channels. Prolonged exposure to PMA (24 h) led to a 60% recovery of the 8-Br-cAMP response, but not of the carbachol- or PDB-provoked secretion. As shown by immunoblotting with PKC-isoenzyme-specific antisera, the recovery of the 8-Br-cAMP response coincided with the down-regulation of PKC alpha, whereas the levels of PKC beta 1 and gamma were unmodified. These results suggest that PKC alpha, but not PKC beta 1 or gamma, is involved in both acute stimulation and chronic inhibition of ion secretion in the HT29cl.19A colonic cell line.

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Chronic treatment by the calcium channel blocker ± PN 200-110 in the rat counteracts the stimulations of pituitary weight, prolactin release and pituitary C-kinase activity induced by a chronic estradiol treatment, in vivo

Acta Endocrinologica ◽

10.1530/acta.0.1220403 ◽

1990 ◽

Vol 122 (3) ◽

pp. 403-408

Author(s):

Ph. Touraine ◽

P. Birman ◽

F. Bai-Grenier ◽

C. Dubray ◽

F. Peillon ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Calcium Channel ◽

Calcium Channel Blocker ◽

Channel Blocker ◽

Plasma Prolactin ◽

Estradiol Treatment ◽

Kinase C ◽

Protein Kinase C Activity

Abstract In order to investigate whether a calcium channel blocker could modulate the protein kinase C activity in normal and estradiol pretreated rat pituitary, female Wistar rats were treated or not (controls) with ± PN 200-110 (3 mg · kg−1 · day−1, sc) for 8 days or with estradiol cervical implants for 8 or 15 days, alone or in combination with PN 200-110 the last 8 days. Estradiol treatment induced a significant increase in plasma prolactin levels and pituitary weight. PN 200-110 administered to normal rats did not modify these parameters, whereas it reduced the effects of the 15 days estradiol treatment on prolactin levels (53.1 ± 4.9 vs 95.0 ±9.1 μg/l, p<0.0001) and pituitary weight (19.9 ± 0.4 vs 23.0 ± 0.6 mg, p <0.001), to values statistically comparable to those measured after 8 days of estradiol treatment. PN 200-110 alone did not induce any change in protein kinase C activity as compared with controls. In contrast, PN 200-110 treatment significantly counteracted the large increase in soluble activity and the decrease in the particulate one induced by estradiol between day 8 and day 15. We conclude that PN 200-110 opposed the stimulatory effects of chronic in vivo estradiol treatment on plasma prolactin levels and pituitary weight and that this regulation was related to a concomitant modulation of the protein kinase C activity.

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