TY - JOUR
T1 - Protein kinase C isoforms in human aortic smooth muscle cells
AU - Grange, J. J.
AU - Baca-Regen, L. M.
AU - Nollendorfs, A. J.
AU - Persidsky, Y.
AU - Sudan, D. L.
AU - Baxter, B. T.
AU - Alexander, J.
N1 - Funding Information:
Supported by a grant from the Nebraska Affiliate of the American Heart Association to J. J. Grange and a grant from the Pacific Vascular Foundation and the VA merit review to B. T. Baxter.
PY - 1998
Y1 - 1998
N2 - Purpose: 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 α, β(I), β(II), δ, ε, γ and ζ. 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 α and β1 were detected primarily in the soluble fraction, translocating to the particulate fraction with TPA stimulation (p < 0.0001). The isoforms β(II), δ, and ε were found primarily in the particulate fraction and did not translocate. Immunofluorescent staining confirmed these locations. Neither γ or ζ 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.
AB - Purpose: 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 α, β(I), β(II), δ, ε, γ and ζ. 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 α and β1 were detected primarily in the soluble fraction, translocating to the particulate fraction with TPA stimulation (p < 0.0001). The isoforms β(II), δ, and ε were found primarily in the particulate fraction and did not translocate. Immunofluorescent staining confirmed these locations. Neither γ or ζ 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.
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U2 - 10.1016/S0741-5214(98)70273-3
DO - 10.1016/S0741-5214(98)70273-3
M3 - Article
C2 - 9620145
AN - SCOPUS:0031822470
VL - 27
SP - 919
EP - 927
JO - Journal of Vascular Surgery
JF - Journal of Vascular Surgery
SN - 0741-5214
IS - 5
ER -