Specific inhibition of phorbol ester and DiC8-induced histamine release from human basophils by the protein kinase C inhibitors, H-7 and H-9

The aim of this study was to determine whether the increase in cytosolic free Ca2+ concentration ([Ca2+]i) in response to antigen (aggregated ovalbumin) on IgE-primed 2H3 cells was sufficient to account for exocytosis. When the [Ca2+]i responses to antigen and the Ca2+ ionophore A23187 were compared, A23187 was much less effective at releasing histamine at equivalent [Ca2+]i increases, and little or no stimulated histamine release occurred with A23187 concentrations that matched the [Ca2+]i response to antigen concentrations that stimulated maximal histamine release. The [Ca2+]i response to antigen is not, therefore, sufficient to account for exocytosis, although extracellular Ca2+ is necessary to initiate both the [Ca2+]i response and histamine release: the antigen must generate an additional, unidentified, signal that is required for exocytosis. To determine whether this signal was the activation of protein kinase C, the effects of the phorbol ester 12-0-tetradecanoyl phorbol 13-acetate (TPA) on the responses to antigen were examined. TPA blocked the antigen-induced [Ca2+]i response and the release of inositol phosphates but had little effect on histamine release and did not stimulate exocytosis by itself. The unidentified signal from the antigen is therefore distinct from the activation of protein kinase C and is generated independently of the [Ca2+]i response or the release of inositol phosphates. Taken together with other data that imply that there is very little activation of protein kinase C by antigen when the rate of histamine release is maximal, it is concluded that the normal exocytotic response to antigen requires the synergistic action of the [Ca2+]i signal together with an unidentified signal that is not mediated by protein kinase C.

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The effect of protein kinase C activation on muscarinic-M3- and K+-evoked release of [3H]noradrenaline and increases in intracellular Ca2+ in human neuroblastoma SH-SY5Y cells

Biochemical Journal ◽

10.1042/bj2820645 ◽

1992 ◽

Vol 282 (3) ◽

pp. 645-650 ◽

Cited By ~ 29

Author(s):

N P Murphy ◽

J G McCormack ◽

S G Ball ◽

P F T Vaughan

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Phorbol Ester ◽

Polymyxin B ◽

Muscarinic Agonist ◽

Muscarinic Agonists ◽

Human Neuroblastoma ◽

Protein Kinase C Inhibitors ◽

Kinase C ◽

M3 Muscarinic Receptor

Short-term pretreatment (9 min) with the phorbol ester 12-myristate 13-acetate (PMA) alone had no effect on the basal release of [3H]noradrenaline ([3H]NA), but enhanced K+ (100 mM)-, acetylcholine (0.1 mM)-, carbachol (1 mM)-, muscarine (1 mM)- and arecoline (1 mM)-evoked release by 2.3-, 6.4-, 3.0-, 2.0- and 2.0-fold respectively in SH-SY5Y cells. Maximum effects of PMA were observed after a 10 min preincubation at a concentration of 0.1 microM. There was a 4-fold decrease in the EC50 values (concentration required for 50% of maximal stimulation) observed for carbachol- and acetylcholine-evoked release of [3H]NA in the presence of PMA. The inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate did not alter the K(+)- or carbachol-evoked release of [3H]NA. The enhancement of release in the presence of PMA was more potently inhibited by the protein kinase C inhibitors RO 31-7549 [concentration required for 50% inhibition (IC50) = 0.18 microM/bd and RO 31-8220 (IC50 = 0.56 microM) than by either polymyxin-B or H-7. Furthermore, in the absence of PMA, both K(+)- and carbachol-evoked release was inhibited by these antagonists. Atropine, hexahydro-sila-difenidol and pirenzepine antagonized the PMA-enhanced carbachol-evoked release of [3H]NA, with Ki values of 2.75 +/- 0.25 nM, 2.6 +/- 0.64 nM and 294 +/- 17 nM respectively. These values were consistent with the coupling of an M3 muscarinic receptor to the release of [3H]NA in SH-SY5Y cells. Whereas pretreatment with PMA (5 min) enhanced M3-evoked release of [3H]NA, it decreased the muscarinic-agonist-evoked initial peak (greater than 85%) and plateau phase in intracellular Ca2+. These results suggest that noradrenaline release evoked by muscarinic agonists was triggered not only by relatively small changes in Ca2+ but also by activation of protein kinase C.

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Phorbol ester potentiation of canine pulmonary vasoreactivity to histamine

Journal of Applied Physiology ◽

10.1152/jappl.1995.79.1.102 ◽

1995 ◽

Vol 79 (1) ◽

pp. 102-106 ◽

Cited By ~ 4

Author(s):

S. A. Barman ◽

S. R. Ikeda

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Phorbol Ester ◽

Phorbol Esters ◽

Vascular Occlusion ◽

Protein Kinase C Inhibitors ◽

Vascular Compliance ◽

Kinase C ◽

Voltage Dependent ◽

Middle Compartment

The effect of phorbol myristate acetate (PMA) on canine pulmonary vasoreactivity to histamine was determined in the isolated blood-perfused dog lung. Pulmonary vascular resistances and compliances were measured by using vascular occlusion techniques. Histamine (10(-5) M) significantly increased postcapillary resistance by venoconstriction and significantly attenuated total vascular compliance by decreasing large-vessel compliance and middle-compartment compliance. Pretreatment with the phorbol ester PMA (10(-7) M) significantly potentiated the vasoactive response to histamine and elicited an edemagenic effect in the isolated dog lung through modulation of the histaminergic vasoconstrictor effect on precapillary resistance, postcapillary resistance, and pulmonary vascular compliance. Pretreatment with the protein kinase C inhibitors staurosporine (10(-7) M) and calphostin C (10(-6) M) and the dihydropyridine Ca2+ channel blocker nifedipine (10(-5) M) significantly attenuated the effect of PMA on histaminergic-mediated vasoconstriction. The results of this study indicate that phorbol esters may exert their effect on canine pulmonary vasoreactivity predominantly through activation of protein kinase C and influx of Ca2+ through voltage-dependent Ca2+ channels.

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Protein kinase C inhibitors reduce phorbol ester-induced resistance to methotrexate in Chinese hamster ovary cells

Biochemical Pharmacology ◽

10.1016/0006-2952(95)00147-r ◽

1995 ◽

Vol 50 (3) ◽

pp. 337-346 ◽

Cited By ~ 6

Author(s):

Véronique Noé ◽

Carlos J. Ciudad

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Induced Resistance ◽

Phorbol Ester ◽

Chinese Hamster Ovary ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Ovary Cells ◽

Protein Kinase C Inhibitors ◽

Kinase C

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Expression of Protein Kinase C Isozymes in Human Basophils: Regulation by Physiological and Nonphysiological Stimuli

Blood ◽

10.1182/blood.v92.4.1206.416k01_1206_1218 ◽

1998 ◽

Vol 92 (4) ◽

pp. 1206-1218 ◽

Cited By ~ 1

Author(s):

Katsushi Miura ◽

Donald W. MacGlashan Jr

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Histamine Release ◽

Membrane Fraction ◽

Resting Cells ◽

Cytosol Fraction ◽

Ige Antibody ◽

Kinase C ◽

Pkc Isozymes ◽

Human Basophils

The expression of protein kinase C (PKC) isozymes in human basophils and the regulation of PKC isozymes during basophil activation by phorbol 12-myristate 13-acetate (PMA) ± ionomycin, f-met-leu-phe (FMLP), and anti-IgE antibody were examined. In human basophils (> 98% purity), PKCβΙ, βΙΙ, δ, and  were expressed, PKC was difficult to detect, and PKCγ and η were undetectable. In unstimulated basophils, PKCβI and βII were found primarily in the cytosol fraction (95% ± 3% of total and 98% ± 1%, respectively). Within 5 minutes of stimulation with PMA (100 ng/mL), both PKCβI and βII were translocated to the membrane fraction (85% ± 4% and 83% ± 6%, respectively). In resting cells, 48% ± 3% and 61% ± 10% of PKCδ and , respectively, existed in the membrane fraction. Within 1 minute of stimulation with PMA, 90% ± 6% of PKC was found in the membrane fraction, however, no translocation of PKCδ was apparent. Stimulation with FMLP caused modest translocation (≈20%) of all PKC isozymes by 1 minute, whereas stimulation with anti-IgE antibody led to no detectable changes in PKC location throughout a 15-minute period of measurement. However, concentrations of PMA and ionomycin that alone caused no PKC translocation and little histamine release, together caused significant histamine release but no apparent PKC translocation. Studies with bis-indolylmaleimide analogs showed inhibition of PMA-induced, but not anti–IgE-induced, histamine release. These pharmacological studies suggest that PKC does not play a prodegranulatory role in human basophil IgE-mediated secretion. © 1998 by The American Society of Hematology.

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Expression of Protein Kinase C Isozymes in Human Basophils: Regulation by Physiological and Nonphysiological Stimuli

Blood ◽

10.1182/blood.v92.4.1206 ◽

1998 ◽

Vol 92 (4) ◽

pp. 1206-1218 ◽

Cited By ~ 26

Author(s):

Katsushi Miura ◽

Donald W. MacGlashan Jr

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Histamine Release ◽

Membrane Fraction ◽

Resting Cells ◽

Cytosol Fraction ◽

Ige Antibody ◽

Kinase C ◽

Pkc Isozymes ◽

Human Basophils

Abstract The expression of protein kinase C (PKC) isozymes in human basophils and the regulation of PKC isozymes during basophil activation by phorbol 12-myristate 13-acetate (PMA) ± ionomycin, f-met-leu-phe (FMLP), and anti-IgE antibody were examined. In human basophils (> 98% purity), PKCβΙ, βΙΙ, δ, and  were expressed, PKC was difficult to detect, and PKCγ and η were undetectable. In unstimulated basophils, PKCβI and βII were found primarily in the cytosol fraction (95% ± 3% of total and 98% ± 1%, respectively). Within 5 minutes of stimulation with PMA (100 ng/mL), both PKCβI and βII were translocated to the membrane fraction (85% ± 4% and 83% ± 6%, respectively). In resting cells, 48% ± 3% and 61% ± 10% of PKCδ and , respectively, existed in the membrane fraction. Within 1 minute of stimulation with PMA, 90% ± 6% of PKC was found in the membrane fraction, however, no translocation of PKCδ was apparent. Stimulation with FMLP caused modest translocation (≈20%) of all PKC isozymes by 1 minute, whereas stimulation with anti-IgE antibody led to no detectable changes in PKC location throughout a 15-minute period of measurement. However, concentrations of PMA and ionomycin that alone caused no PKC translocation and little histamine release, together caused significant histamine release but no apparent PKC translocation. Studies with bis-indolylmaleimide analogs showed inhibition of PMA-induced, but not anti–IgE-induced, histamine release. These pharmacological studies suggest that PKC does not play a prodegranulatory role in human basophil IgE-mediated secretion. © 1998 by The American Society of Hematology.

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