An improved permeabilization protocol for the introduction of peptides into cardiac myocytes. Application to protein kinase C research.
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.
Johnson
J A
JA
Department of Molecular Pharmacology, Stanford University School of Medicine, CA 94305-5332, USA.
Gray
M O
MO
Karliner
J S
JS
Chen
C H
CH
Mochly-Rosen
D
D
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
D010455:Peptides; D012503:Saponins; D011493:Protein Kinase C