Signal transduction events in human basophils. A comparative study of the role of protein kinase C in basophils activated by anti-IgE antibody and formyl-methionyl-leucyl-phenylalanine.
We have compared the transmembrane signals generated in human basophils by two distinct stimuli, anti-IgE antibody and FMLP (f-met peptide). Although both stimuli resulted in the activation of protein kinase C (PKC) and an increase in intracellular free calcium, there were substantial differences between the two which suggested that distinct signal transduction mechanisms were operating. We have confirmed an earlier observation that the cross-linking of IgE led to an increase in membrane PKC activity with no apparent concomitant loss of cytosolic PKC and established that in contrast, the univalent stimulus, f-met peptide, resulted in the canonical translocation of cytosolic PKC to the membrane. Furthermore, unlike anti-IgE-stimulated basophils, there was no clear relationship between the increase in PKC activity and the subsequent release of histamine. Two PKC inhibitors, staurosporine (0.1 to 1 nM) and sphingosine (25 to 50 microM), inhibited anti-IgE induced release, yet, potentiated the release of mediators after a challenge with 1 microM f-met peptide. Both stimuli led to an increase in the intracellular Ca2+ levels that correlated well with the release of histamine, however, the anti-IgE-induced responses were typically only 50% of those required to give equivalent histamine release when f-met peptide initiated release. Pharmacologic evidence suggested that the up-regulation of PKC was required for a full IgE-mediated Ca2+ response and that PKC contributed to the elevated Ca2+ levels that persist for up to 15 min after the addition of anti-IgE. In contrast, the PKC inhibitor, staurosporine, did not affect the initial increase in Ca2+ after the addition of f-met peptide but reduced the rate at which Ca2+ was removed from the cytosol. Experiments with the phorbol ester, PMA, suggested that substantial degranulation can occur in the absence of any increase in intracellular Ca2+. The addition of 10 ng/ml PMA 10 min before the addition of f-met peptide did not affect the magnitude of the initial Ca2+ transient but increased the rate at which Ca2+ levels returned to a stable baseline. Similar pretreatment with PMA almost completely abolished the anti-IgE antibody-induced Ca2+ response. These experiments, together with other previous data, suggest that the activation of PKC is a prodegranulatory component of the IgE-mediated signal transduction pathway, yet serves principally to modulate the Ca2+ signal when f-met peptide initiates release.
Johns Hopkins Asthma and Allergy Center, Baltimore, MD 21224.
J A
JA
Warner
D W
DW
MacGlashan
D000470:Alkaloids / Q000494:pharmacology; D000888:Antibodies, Anti-Idiotypic; D001491:Basophils / Q000201:enzymology / Q000502:physiology*; D002118:Calcium / Q000502:physiology; D015550:Cell Degranulation / Q000187:drug effects; D006636:Histamine Release / Q000187:drug effects; D006801:Humans; D007073:Immunoglobulin E / Q000502:physiology*; D066298:In Vitro Techniques; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology*; D011940:Receptor Aggregation; D013189:SRS-A / Q000378:metabolism; D015398:Signal Transduction; D013110:Sphingosine / Q000494:pharmacology; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000470:Alkaloids; D000888:Antibodies, Anti-Idiotypic; D013189:SRS-A; D007073:Immunoglobulin E; D009240:N-Formylmethionine Leucyl-Phenylalanine; D011493:Protein Kinase C; D019311:Staurosporine; D013110:Sphingosine; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium