Abstract
Antimicrobial peptides are produced by nearly all organisms. Certain antimicrobial
peptides from multicellular animals also kill a variety of tumour cells at concentrations not
affecting normal eukaryotic cells. Recently, it was reported that also Plantaricin A (PlnA),
which is a peptide with antibacterial activity produced by Lactobacillus plantarum, may kill
eucaryotic cells. It was shown that PlnA permeabilizes cancerous rat pituitary cells (GH4
cells), whereas normal rat anterior pituitary cells are resistant to the peptide. The same study
also showed that exposure of PlnA to the inside of the membrane, did not lead to
permeabilization.
This thesis involved three tasks. The first was to examine whether PlnA differentiates
between various normal cells, and the second task was to elucidate if the membrane
permeabilizing effect of PlnA is restricted to cancerous cells. An examination of the
permeabilizing effect of PlnA on the outer and inner membrane leaflets was the final task. In
order to examine these questions, we have studied primary cultures of rat liver cells
(hepatocytes, endothelial- and Kupffer cells), primary culture of rat kidney cells and two
epithelial cell lines of primate kidney origin (Vero cells from green monkey and human
CAKI-2 cells). The Vero cell line is derived from normal cells, whereas the CAKI-2 cell line
is derived from a cancerous tumour. The membrane effects were studied by means of patch
clamp recordings and microfluorometric (fura-2) monitoring of the cytosolic concentrations
of Ca2+ ([Ca2+]i) and fluorochrome.
In all of the cells an exposure to 100 500 μM PlnA induced a nearly instant
permeabilization of the membrane, indicated by the following criteria: increased membrane
conductance, membrane depolarization, increased [Ca2+]i, and diffusional loss of
fluorochrome from the cytosol. When exposed to 100 μM PlnA, none of the inside-out
patches from Vero cells were permeabilized. On the contrary, more than 80 % of the outsideout
patches were permeabilized at 10 μM PlnA. We thus conclude that the permeabilizing
effect of PlnA is not restricted to neither of the normal tested cell types nor the cancerous
cells, and that PlnA differentiate between the inner and outer membrane leaflet.