Involvement of calcium in modulation of neutrophil function by phorbol esters that activate protein kinase C isotypes and related enzymes.
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)
Merritt
J E
JE
SmithKline Beecham Pharmaceuticals Ltd., The Frythe, Welwyn, Herts, U.K.
Moores
K E
KE
Evans
A T
AT
Sharma
P
P
Evans
F J
FJ
MacPhee
C H
CH
D002118:Calcium / Q000378:metabolism*; D004224:Diterpenes / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005609:Free Radicals; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D015759:Ionomycin / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism*; D008345:Manganese / Q000378:metabolism; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000502:physiology*; D010100:Oxygen / Q000378:metabolism; D010703:Phorbol Esters / Q000494:pharmacology*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D004224:Diterpenes; D005609:Free Radicals; D007093:Imidazoles; D007527:Isoenzymes; D010703:Phorbol Esters; D010975:Platelet Aggregation Inhibitors; D008345:Manganese; D015759:Ionomycin; C029704:12-deoxyphorbolphenylacetate; D009240:N-Formylmethionine Leucyl-Phenylalanine; C032016:sapintoxin A; C024353:resiniferatoxin; D011493:Protein Kinase C; C063159:1-(2-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenylethyl)-1H-imidazole; D013755:Tetradecanoylphorbol Acetate; D010100:Oxygen; D002118:Calcium