title: An improved permeabilization protocol for the introduction of peptides into cardiac myocytes. Application to protein kinase C research.
abstract: We have developed an improved, less disruptive procedure for the transient permeabilization of neonatal cardiac myocytes using saponin. The method allows delivery of peptides to a high percentage of cells in culture without effects on long-term cell viability. Permeation was confirmed microscopically by cellular uptake of a fluorescently labeled peptide and biochemically by uptake of 125I-labeled calmodulin and a 20-kD protein kinase C epsilon fragment into the cells. The intracellular molar concentration of the introduced peptide was approximately 10% of that applied outside. We found no significant effects of permeabilization on spontaneous, phorbol ester-modulated, or norepinephrine-modulated contraction rates. Similarly, the expression of c-fos mRNA (measured 30 minutes after permeabilization) and the incorporation of [-14C]phenylalanine following agonist stimulation (measured 3 days after permeabilization) were not altered by saponin permeabilization. Finally, permeabilization of cells in the presence of a protein kinase C pseudosubstrate peptide, but not a control peptide, inhibited phorbol ester-induced [14C]phenylalanine incorporation into proteins by 80%. Our results demonstrate a methodology for the introduction of peptides into neonatal cardiac myocytes that allows study of their actions without substantial compromises in cell integrity.
authors.affiliation: Department of Molecular Pharmacology, Stanford University School of Medicine, CA 94305-5332, USA.
authors.forename: J A
authors.initials: JA
authors.lastname: Johnson
authors.forename: M O
authors.initials: MO
authors.lastname: Gray
authors.forename: J S
authors.initials: JS
authors.lastname: Karliner
authors.forename: C H
authors.initials: CH
authors.lastname: Chen
authors.forename: D
authors.initials: D
authors.lastname: Mochly-Rosen
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D006321:Heart / Q000502:physiology*; D009200:Myocardial Contraction / Q000502:physiology*; D010455:Peptides / Q000502:physiology*; D011493:Protein Kinase C / Q000032:analysis / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012503:Saponins
chemical_list: D010455:Peptides; D012503:Saponins; D011493:Protein Kinase C