Protein kinase C isoforms in human aortic smooth muscle cells.
OBJECTIVE
To identify the protein kinase C (PKC) isoforms in human arterial smooth muscle cells (SMC) and define their subcellular location in the resting state and in response to the PKC activator, 12-O-tetradecanoylphorbol 13-acetate (TPA).


METHODS
Arterial SMC cultures established from transplant donor aorta were treated with 100 nM TPA or control media, then mechanically lysed. PKC from the soluble and particulate fraction were separated by centrifugation, and protein normalized immunoblots were performed with antibodies to the PKC isoforms alpha, betaI, betaII, delta, epsilon, gamma and zeta. Bands were detected by enhanced chemiluminescence and analyzed densitometrically, with results expressed as the mean percentage of each fraction +/- SEM. Translocation was defined as a significant (p < 0.05) change in the particulate fraction for each isoform. Immunofluorescent staining of cultured SMC visualized the resting location and stimulated translocation of each isoform.


RESULTS
Isoforms alpha and betaI were detected primarily in the soluble fraction, translocating to the particulate fraction with TPA stimulation (p < 0.0001). The isoforms betaII, delta, and epsilon were found primarily in the particulate fraction and did not translocate. Immunofluorescent staining confirmed these locations. Neither gamma or zeta were detected in these SMC.


CONCLUSIONS
The PKC isoforms expressed in human arterial SMC differ from those reported in animal models. Their specific locations and response to stimulation suggest unique functions in cellular regulation and provide the groundwork for further investigation into their role in the development of vascular disease and regulation of matrix metabolism.
Grange
J J
JJ
Department of Surgery, University of Nebraska Medical Center, Omaha 68198-3280, USA.
Baca-Regen
L M
LM
Nollendorfs
A J
AJ
Persidsky
Y
Y
Sudan
D L
DL
Baxter
B T
BT
D000906:Antibodies; D001011:Aorta / Q000166:cytology / Q000201:enzymology*; D002469:Cell Separation; D002478:Cells, Cultured; D004396:Coloring Agents; D003720:Densitometry; D004789:Enzyme Activation; D005455:Fluorescent Antibody Technique; D006801:Humans; D015151:Immunoblotting; D007527:Isoenzymes / Q000032:analysis* / Q000187:drug effects / Q000648:ultrastructure; D008163:Luminescent Measurements; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000201:enzymology*; D011493:Protein Kinase C / Q000032:analysis* / Q000187:drug effects / Q000648:ultrastructure; D012995:Solubility; D013347:Subcellular Fractions / Q000201:enzymology / Q000648:ultrastructure; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000906:Antibodies; D004396:Coloring Agents; D007527:Isoenzymes; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate