Contrasting effects of the protein kinase C inhibitor staurosporine on the interleukin-1 and phorbol ester activation pathways in the EL4-6.1 thymoma cell line

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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DIACYLGLYCEROL AND PHORBOL ESTER REDUCE AEQUORIN-INDICATED [Ca++] ELEVATIONS INDUCED BY THROMBIN OR ADP

10.1055/s-0038-1644503 ◽

1987 ◽

Author(s):

J A Ware ◽

M Smith ◽

E W Salzman

Keyword(s):

Protein Kinase C ◽

Platelet Aggregation ◽

Protein Kinase ◽

Phorbol Ester ◽

Time Dependent ◽

Dependent Manner ◽

Kinase C ◽

Protein Kinase C Activators ◽

Protein Kinase C Inhibitor ◽

Ca Homeostasis

Platelet aggregation and secretion induced by phorbol ester (PMA) or diacylglycerol (DAG) are preceded by an increase in [Ca++] that is detected byaequorin, but not by quin2, fura-2, or indo-1, suggesting that these indicatorsreflect different aspects of Ca++ homeostasis, possibly different functional Ca++ pools. Addition of two conventional agonists in subthreold concentrations synergistically enhances the [Ca++] rise and aggregation.However, if PMA or DAG is the first agonist the subsequent quin2-indicated [Ca++] rise after thrombin is reduced.Whether aequorin-indicated [Ca++] is similarly affected is unknown. We studied gel-filtered platelets loaded with aequorin or a fluorophore and added PMA, DAG, thrombin or ADP, alone or in combination. Either PMA or DAG alone caused a concentration-dependent increase in [Ca++] detectable with aequorin but not with the fluorophores; simultaneous addition of thrombin or ADP with DAG or PMA produced a larger [Ca++] rise than either alone. However, addition of DAG or PMA as a first agonist reduced subsequent aequorin-indicated [Ca++] rises following thrombin or ADP in a concentration and time-dependent manner. Inhibition of ADP or thrombin-induced [Ca++] rise was not always accompanied by inhibition of aggregation or secretion. Combination of subthreshold concentrations of ADP and thrombin produced an enhanced [Ca++] rise and aggregation. However, this synergistic effect was inhibited by preincubation with DAG or PMA. Neither this effect nor DAG-induced [Ca++] rise was inhibited by the protein kinase C inhibitor H-7. In genera^ preincubation of platelets with an agonist enhances Ca rise and aggregation in response to a second agonist; in contrasl protein kinase C activators, which themselves elevate [Ca++] as shown by aequorin, inhibit aequorin-indicated Ca rises after ADP or thrombin, and limit synergism between these two agonists.

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Ca2+ channel ligand sensitive responses to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate in vascular smooth muscle

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y86-251 ◽

1986 ◽

Vol 64 (12) ◽

pp. 1489-1496 ◽

Cited By ~ 10

Author(s):

X. Y. Wei ◽

D. J. Triggle

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Phorbol Ester ◽

Rat Aorta ◽

Bay K 8644 ◽

Alternative Possibilities ◽

Kinase C ◽

Protein Kinase C Inhibitor ◽

Physiologic Saline ◽

Rat Tail

The action of a tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), on isolated rat aortic and tail artery strips has been characterized. TPA (10−9 – 10−7 M) produced a graded contraction developing maximum tension over 30–40 min. The contraction was irreversible and was not relaxed by prolonged washing with physiologic saline. Relaxation occurred upon washing with Ca2+-free saline but readdition of Ca2+ restored response. TPA was without significant effect in rat tail arteries in physiologic saline but produced responses in saline containing elevated K+ (15 mM). The protein kinase C inhibitor, CP-46,665-1 (4-aminomethyl-1-[2,3-(di-n-decyloxy)n-propyl]-4-phenylpiperidine dihydrochloride) (5 × 10−5 M), blocked the response to TPA but was without effect on responses to Bay K 8644 (2,6-dimethyl-3-carbomethoxy-5-nitro-4-(2-trifluoromethylphenyl) 1,4-dihydropyridine), KCl, phenylephrine, and B-HT 920 (6-allyl-2-amino-5,6,7,8-tetrahydro-4H-thiazolo[4,5-d]azepin dihydrochloride). The calcium channel antagonist nifedipine and its analogue, 2,6-dimethyl-3,5-dicarbomethoxy-4-(3-cyanophenyl)-1,4-dihydropyridine, inhibited TPA responses with IC50 values of 9.28 × 10−9 and 1.96 × 10−7 M, respectively. Responses to Bay K 8644 in rat aorta were maximum in the presence of elevated KCl (10 mM), but TPA at concentrations of 10−9 and 3 × 10−9 M potentiated responses to Bay K 8644 in physiologic saline to levels approximating those in elevated K+ saline. TPA similarly potentiated responses to Ca2+ in Ca2+-free solution. In the presence of TPA, 10−8 and 3 × 10−8 M, responses to Ca2+ in nondepolarizing saline were potentiated to levels seen under depolarizing conditions. The present results suggest a relationship between protein kinase C and Ca2+ channel activation. However, alternative possibilities, including enhancement of the Ca2+ sensitivity of the contractile apparatus, may also contribute to the observed effects.

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