Nuclear Translocation of PKA Catalytic Subunit

Immunocytochemical investigations showed that in untreated pinealocytes the regulatory and catalytic subunits are exclusively located in the cytosol. Under NE-treatment a certain amount of the catalytic subunits entered the nucleus (Figure 7a) whereas the regulatory subunits remained in the cytosol (data not shown). NE-induced nuclear translocation of the PKA catalytic subunit could also be shown by immuno-blotting of nuclear extracts (Figure 7b). The nuclear entry of the PKA catalytic subunit is regulated by a P-adrenergic cAMP pathway, since NE, as well as isoproterenol and

Figure 6. Immunochemical detection of the cyclic AMP-dependent protein kinase subunits C (a/b), RIa (c/d), Rlla (e/f) and RIip (g/h) in rat pineal glands obtained ex vivo at different times under 12h L: 12 h D. a, c, e and g show representative original immunoblots with the timepoints indicated on top of each blot and the size markers on the right side. b, d, f and h show semi-quantifications of a total of 3 to 4 such experiments. The open bars under each graph represents the light phase, the black bars the dark phase. The error bars represent means ± SEM.

Figure 6. Immunochemical detection of the cyclic AMP-dependent protein kinase subunits C (a/b), RIa (c/d), Rlla (e/f) and RIip (g/h) in rat pineal glands obtained ex vivo at different times under 12h L: 12 h D. a, c, e and g show representative original immunoblots with the timepoints indicated on top of each blot and the size markers on the right side. b, d, f and h show semi-quantifications of a total of 3 to 4 such experiments. The open bars under each graph represents the light phase, the black bars the dark phase. The error bars represent means ± SEM.

Co lpMNE(lh)

Figure 7. Translocation of the catalytic subunit of PKA (cPKA) into the nucleus after norepinephrine-stimulation for one hour. A The image on the left side shows cPKA-like-immunoreactivity in untreated rat pinealocytes, the image on the right side shows cPKA-like-immunoreactivity in rat pinealocytes treated with NE for one hour. The treated pinealocytes contain more immunoreactive material in their nuclei than the untreated controls. B Percentage of nuclear immunoreactivity for cPKA in extracts of pineal organs treated with NE for one hour or left untreated (Co). Shown are means ± SEM (t-test, p < 0.05; N = 4).

cAMP analogs induced nuclear translocation of cPKA to a very similar extent (Maronde et al., unpublished observations). These results suggest that nuclear translocation of catalytic subunits is an essential step for the induction of CREB phosphory-lation in rat pinealocytes.

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