Enhancement of intracellular concentration and biological activity of PNA after conjugation with a cell-penetrating synthetic model peptide.
In order to evaluate the ability of the cell-penetrating alpha-helical amphipathic model peptide KLALKLALKALKAALKLA-NH(2) (MAP) to deliver peptide nucleic acids (PNAs) into mammalian cells, MAP was covalently linked to the 12-mer PNA 5'-GGAGCAGGAAAG-3' directed against the mRNA of the nociceptin/orphanin FQ receptor. The cellular uptake of both the naked PNA and its MAP-conjugate was studied by means of capillary electrophoresis combined with laser-induced fluorescence detection, confocal laser scanning microscopy and fluorescence-activated cell sorting. Incubation with the fluorescein-labelled PNA-peptide conjugate led to three- and eightfold higher intracellular concentrations in neonatal rat cardiomyocytes and CHO cells, respectively, than found after exposure of the cells to the naked PNA. Correspondingly, pretreatment of spontaneously-beating neonatal rat cardiomyocytes with the PNA-peptide conjugate and the naked PNA slowed down the positive chronotropic effect elicited by the neuropeptide nociceptin by 10- and twofold, respectively. The main reasons for the higher bioavailability of the PNA-peptide conjugate were found to be a more rapid cellular uptake in combination with a lowered re-export and resistance against influences of serum.
Oehlke
Johannes
J
Institute of Molecular Pharmacology, Berlin, Germany. oehlke@fmp-berlin.de
Wallukat
Gerd
G
Wolf
Yvonne
Y
Ehrlich
Angelika
A
Wiesner
Burkhard
B
Berger
Hartmut
H
Bienert
Michael
M
D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D001692:Biological Transport / Q000502:physiology; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D004705:Endocytosis / Q000502:physiology*; D006801:Humans; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D018847:Opioid Peptides / Q000494:pharmacology; D020135:Peptide Nucleic Acids / Q000378:metabolism*; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011957:Receptors, Opioid / Q000235:genetics; D000094942:Nociceptin Receptor; D000097629:Nociceptin
D018847:Opioid Peptides; D020135:Peptide Nucleic Acids; D010455:Peptides; D012333:RNA, Messenger; D011957:Receptors, Opioid; D000094942:Nociceptin Receptor; D000097629:Nociceptin