No abstract

PKC has been shown to modulate the binding of ligand to receptors (e.g. transferrin and epidermal growth factor), and we suggest that at least part of this effect is due to receptor internalisation following stimulation of endocytosis by PKC.To better understand the role of PKC in the regulation of endocytosis we have investigated the outcome of activation of endogenous PKC with phorbol ester in the murine neuroblastoma cell line (N2A). Alterations in endocytosis have been established through use of the membrane specific lipophilic styrl dye FM 4-64 and confocal microscopy. Cells were pre-loaded with FM 4-64 at < 4"C, in order to inhibit dye internalisation, and then incubated at 37°C for varying times, fixed in glutaraldehyde and viewed.Activation of PKC in N2A cells with phorbol ester results in a rapid increase in the level of dye internalisation. Cells treated for 1 min displayed a punctate dye distribution spread throughout the cytoplasm while untreated cells maintain high levels of fluorescence at the membrane and little dye internalisation. Cells treated for 3min show fewer but larger areas of dye concentration in the cytoplasm. Untreated cells still displayed membrane localisation of dye although an increase in internalisation was apparent. Treated and untreated cells showed similar patterns of dye distribution after 30min incubation at 37°C.These findings indicate that PKC activation increased membrane internalisation in N2A cells, suggesting a role for PKC in the upregulation of the endocytic rate. Ongoing work, using electron microscopy and ligand binding studies, will further define the nature of this relationship 52 Expression and localisation of protein kinase C isotypes in equine eosinophils. Activation of equine eosinophils by phorbol esters suggests that protein kinase C (PKC) may regulate the function of these cells (Foster and Cunningham 1997, Vet. Immunol. Immunopathol. 59: 225-237). PKC consists of at least 12 isotypes and as a prerequisite to studying isotype specific effects we have established the expression pattern of PKCs in equine eosinophils.A number of PKC isotypes were found to be expressed in equine eosinophils including conventional a, PI, 011, (but not y), novel 6, E, (but not q or e), and atypical <, I (but not A). The identified isotypes were, unusually, found mainly in the particulate fraction, in contrast to the cytosolic and membrane sites for PKC in human eosinophils (Evans ef al. 1996, Am. J.Respir. Crit. Care Med. 153: A57) and other cell types as well as our studies on equine neutrophils. Further fractionation showed the PKCs to be in the nucleus. In an attempt to activate and thereby change this unusual PKC localisation, cells were treated with either phorbol ester or Ca2+ ionophore. Neither of these treatments altered the location of assessed PKCs, but both caused a very rapid down-regulation within 20 min. Dephosphorylation is thought to precede down regulation; therefore eosinophils were pre-treated with the protease inhibitor okadaic acid, but no effect was seen on ...

PKC is involved in regulating equine eosinophil adherence and superoxide production. The role of PKCdelta appears to depend upon the stimulus used and response measured.

Phorbol esters, which activate protein kinase C (PKC), stimulate equine eosinophil superoxide production and adherence. After showing that superoxide production could be inhibited by the nonselective PKC inhibitors, staurosporine and bisindolymaleimide I, the PKC isotypes in equine eosinophils were characterized, because evidence suggests that individual isotypes may play distinct roles in regulating eosinophil function. Western blots demonstrated that equine eosinophils expressed PKC α, β, δ, ɛ, ι, and ζ. However, unlike the equine neutrophil, the majority of the PKC was detected in the particulate fraction of the cell. Despite this unusual location, the PKC in equine eosinophils was activatable, suggesting that it is functionally competent. The regulatory role of PKC in equine eosinophils may reflect the association of activity with the particulate fraction and the profile of isotype expression.