Kreatinstoffwechsel in humaner Haut

Various aspects of the Creatine metabolism in human skin were researched within the framework of this study. These aspects are summarised as follows: The existence of cytoplasmic creatine kinase (CK-BB), mitochondrial creatine kinase (CK-mt) as well as a creatine transporter (CT) could be proven in the human skin immune-histologically and by western blot analysis. Furthermore, the localisation of the CK-BB and CK-mt and the functionality of these enzymes could be demonstrated in vitro in primary human keratinocytes. The active uptake of creatine in primary human keratinocytes and primary human dermal fibroblasts was investigated using the CT. Creatine kinase activity was measured in primary, human keratinocytes from old and young donors, and showed a reduction in the activity during the aging process. This reduction in the activity could be thwarted by creatine supplementation as could the inhibition of the creatine kinase activity caused by hydrogen peroxide. Therefore it was demonstrated that creatine exhibits a protective effect against oxidative stress, caused by an increase in reactive oxygen species (ROS) known to occur in the aging process. This protective effect can be explained by the stabilising property of creatine on the CK-mt and thus also on the mitochondrial membrane potential, as the CK-mt appears to be the predominantly occurring isoform in keratinocytes. The key enzymes of creatine synthesis: Guanidinoactate-Methyl-Transferase (GAMT) and Arginine-Glycine-Amidino-Transferase (AGAT) could be demonstrated in human skin, primary human keratinocytes and primary human dermal fibroblasts on mRNA level. Furthermore, the creatine kinase activity could be stimulated after incubation with the creatine synthesis precursor Guanidinoacetate in in vitro tests performed on keratinocytes. The inhibition of the CK-BB and CK-mt in cells of the human keratinocytic cell line HaCat and the human cervical carcinogenic cell line HeLa S3 showed, sometimes-drastic effects on the cells. Particularly the inactivation of the CK-mt leads to distinct cytotoxic effects, decreased proliferation and a reduction in the mitochondrial membrane potential. The results of a loss of the CK-mt can be observed electron microscopically on the basis of a change in mitochondrial membrane. An increase in the apoptotic and necrotic processes can be observed when the least expressed creatine kinase present in each of the cell lines was deactivated. The attained data shows, not only the relevance of the Creatine Kinases for the cell, but also supports the findings to the specialised role of the CK-mt in the stabilisation of the Mitochondrial membrane.