Rottlerin induces calcium influx and protein degradation in cultured lenses independent of effects on protein kinase C delta.
Rottlerin has been widely accepted as a specific inhibitor of protein kinase C delta (PKC delta); however, recent data suggest that the specificity of this compound become a question. Herein, we address this issue using a lens organ culture system, as PKC delta might regulate the gap junction permeability in lens. Interestingly, we found that rottlerin induced the degradation of connexin50 more rapidly than that of PKC delta. Furthermore, comparison of rottlerin with a protonophore, carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP) that shares many characteristics with rottlerin, showed that both rottlerin and FCCP dramatically increased lens weight over time. This increase in lens weight was partially reversed by depletion of extracellular calcium with ethyleneglycoltetraacetic acid (EGTA) or by blocking L-type calcium channels with verapamil, suggesting rottlerin may induce calcium influx. Indeed, the rapid degradation of connexin50 (but not PKC delta) induced by rottlerin and FCCP was blocked by EGTA. In addition, rottlerin and FCCP also induced degradation of connexin46, filensin, vimentin and CP49. In order to determine whether this protein degradation is associated with the decrease of ATP due to uncoupling mitochondria by rottlerin, ATP content in lenses with different treatments were examined. The result indicated that EGTA had no effect on lens ATP content. Taken together, these data suggest that rottlerin, like FCCP, induces calcium influx, leading to protein degradation and cleavage in the lens, and that this effect is unrelated to the inhibition of PKC delta. Thus, extreme caution must be taken when considering use of rottlerin as a PKC delta inhibitor.
Xu
Shang-Zhi
SZ
Department of Neuroscience, University of Minnesota, Minneapolis, MN 55455, USA. xushzh@yahoo.com
D000098:Acetophenones / Q000494:pharmacology*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001578:Benzopyrans / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000187:drug effects / Q000378:metabolism; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D017630:Connexins / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005136:Eye Proteins / Q000187:drug effects / Q000378:metabolism; D007381:Intermediate Filament Proteins / Q000187:drug effects / Q000378:metabolism; D007908:Lens, Crystalline / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008928:Mitochondria / Q000378:metabolism; D009924:Organ Culture Techniques; D009929:Organ Size; D051745:Protein Kinase C-delta / Q000187:drug effects / Q000378:metabolism; D014746:Vimentin / Q000187:drug effects / Q000378:metabolism; D000099264:Connexin 50
D000098:Acetophenones; D000255:Adenosine Triphosphate; D001578:Benzopyrans; D002118:Calcium; D020746:Calcium Channels, L-Type; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D017630:Connexins; D004791:Enzyme Inhibitors; D005136:Eye Proteins; D007381:Intermediate Filament Proteins; D051745:Protein Kinase C-delta; D014746:Vimentin; D000099264:Connexin 50; C082917:GJA3 protein, human; C079098:filensin; C084808:phakinin; C085746:rottlerin