Induction of apoptosis in cultured human retinal pigmented epithelial cells: the effect of protein kinase C activation and inhibition.
The presence of the non-selective protein kinase C (PKC) inhibitors, staurosporine (100 nM) and polymyxin B (100 microM) in cultured human RPE cells for more than 24 h triggers apoptotic death. Apoptosis is characterized by a diminishing number of cells, a labelling of nuclei by the TUNEL method and by observable morphological changes. An inhibitor of PKC and cyclic nucleotide-dependent protein kinases, 1-(5-isoquinolinesulphonyl)-2-methyl piperazine (H-7; 100 microM), was without effect, as was the specific PKC inhibitor, calphostin C (100 nM). The PKC-activating phorbol esters, phorbol-12-myristate-13-acetate (PMA; 1 microM) and phorbol-12,13-dibutyrate (PDB; 1 microM) and the non-tumour-promoting phorbol ester, 4 alpha-PMA (1 microM) were without effect, as was the diacyl glycerol analogue, 1,2-dioctanoyl-snglycerol (DOG; 10 microM). The PKC activators did not attenuate the apoptosis induced by staurosporine or polymyxin B. Furthermore, deprivation of glucose and oxygen (simulated ischemia) for 72 h induced apoptosis: this could be prevented by inclusion of 10% (v/v) foetal bovine serum (FBS) but not by a variety of PKC activators. Six PKC isoenzymes were shown to be present in RPE cells (alpha, beta 1, beta 2, delta, epsilon, E) and only the calcium-dependent cPKC levels changed after treatment with staurosporine or simulated ischaemia. Since only the less selective inhibitors of PKC induced apoptosis, it is suggested that PKC is not involved directly in the induction process of apoptosis in RPE cells. It is possible that the staurosporine and polymyxin B-induced effects of apoptosis in RPE cells are triggered by an unknown kinase-dependent pathway, but whether the 'ischaemia'-induced death is related to this same process remains to be elucidated.
Wood
J P
JP
Nuffield Laboratory of Ophthalmology, University of Oxford, U.K.
Osborne
N N
NN
D000328:Adult; D017209:Apoptosis; D015153:Blotting, Western; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D008875:Middle Aged; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000502:physiology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors* / Q000378:metabolism*
D007527:Isoenzymes; D011493:Protein Kinase C