Protein kinase C distribution and translocation in rat myocardium: Methodological considerations

Introduction: Protein kinase C (PKC) is an important modifier of several cardiovascular phenomena, including cardioprotection, apoptosis, and hypertrophy. Although pharmacological activation of PKC is often assessed by translocation, the effects of isolation procedures on left ventricular (LV) PKC d...

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Published in:Journal of pharmacological and toxicological methods 2005-03, Vol.51 (2), p.129-138
Main Authors: Hunter, J. Craig, Korzick, Donna H.
Format: Article
Language:English
Subjects:
Animals
Biological Transport, Active
Centrifugation
Detergents - pharmacology
Dose-Response Relationship, Drug
Male
Methods
Myocardium - enzymology
Octoxynol - pharmacology
PMA
Polytron
Protein kinase C
Protein Kinase C - metabolism
Rats
Rats, Inbred F344
Rats, Wistar
Tissue Distribution
Triton X-100
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Summary:Introduction: Protein kinase C (PKC) is an important modifier of several cardiovascular phenomena, including cardioprotection, apoptosis, and hypertrophy. Although pharmacological activation of PKC is often assessed by translocation, the effects of isolation procedures on left ventricular (LV) PKC distribution have not been systematically examined. Accordingly, we sought to determine whether homogenization methods (Polytron, glass–glass tissue grinder), detergent selection and concentration, or centrifugation protocols affect PKC (α, ɛ) distribution or phorbol-12-myristate-13-acetate (PMA)-induced translocation. Methods: Hearts of male F344 or Wistar rats were Langendorff perfused with either 100 nM PMA or vehicle, and LV cytosolic and particulate PKC (α, ɛ) distributions were assessed by differential centrifugation and Western blotting. Results: Following 100 000 × g centrifugation of the homogenate, resuspension of the pellet (P 1) in 0.1% sodium dodecyl sulfate (SDS) increased electrophoretic mobility of PKC (α, ɛ) such that PKCɛ comigrated with a nonspecific band. Resuspension of P 1 in Triton X-100 (TX) did not affect mobility but decreased P 1 PKC (α, ɛ) levels in a TX-concentration-dependent manner; however, this decrease was found to be due to differential protein solubilization. Decreased levels of PKC (α, ɛ) were also noted in soluble and P 2 (supernatant of 100 000 × g centrifugation of P 1) fractions due to increased Polytron burst and total homogenization times. Interestingly, the P 2 fraction also revealed Polytron-dependent decreases (47% vs. glass–glass tissue grinder; p
ISSN:1056-8719
1873-488X
DOI:10.1016/j.vascn.2004.10.003