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Cardiovascular Division, Charles A. Dana Research Institute, Boston, MA 02215.
1. The relationship between phenylephrine-induced smooth muscle contraction and the subcellular distribution of protein kinase C (PKC) was investigated. 2. Cell shortening induced by phenylephrine (10(-5) M) was measured in single vascular cells freshly isolated from ferret portal vein and aorta. 3. At various time points during phenylephrine activation, single cells were fixed with paraformaldehyde and the distribution of PKC was imaged in cells labelled with the fluorescent PKC probe 12-(1,3,5,7-tetramethylBODIPY-2-propionyl)phorbol-13-acetate. 4. The PKC probe located to a perinuclear region, the cytosol and surface membrane. The amplitude and time course of the phenylephrine-induced changes in the surface membrane/cytosol fluorescence ratio were measured and compared with the amplitude and time course of phenylephrine-induced cell shortening. 5. In portal vein cells incubated in 1 mM-external Ca2+, phenylephrine caused significant shortening and time-dependent translocation of the PKC probe to the surface membrane, but cell shortening preceded PKC translocation. In Ca2+free solution both cell shortening and translocation of the probe were completely inhibited. 6. Verapamil (3 x 10(-7) M) partially, but significantly, inhibited the magnitude of cell shortening and delayed the onset and time to peak shortening. Translocation of PKC in verapamil preceded or coincided with cell shortening. 7. In aorta cells incubated in 1 mM-extracellular Ca2+, phenylephrine induced significant shortening and time-dependent translocation of the PKC probe. Cell shortening preceded PKC translocation. In Ca(2+)-free solution, shortening was only partially, but significantly, inhibited and PKC translocation preceded the fraction of the shortening response that remained. 8. These data are consistent with a role for PKC in the maintenance of the phenylephrine-induced contraction in both portal vein and aorta. The data also suggest that phenylephrine-induced contraction may involve activation of a Ca(2+)-dependent PKC isoform in ferret portal vein but a Ca(2+)-independent isoform in ferret aorta.
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