On the other hand, the divergent effects observed with chelerythrine and calphostin C on ERK1/2 phosphorylation and withdrawal behaviour could be due to the inhibition of protein kinase C activity at either the catalytic or the regulatory site with chelerythrine or calphostin C respectively. In conclusion, the present data show an activation of CREB in the CRF neurons of the PVN during morphine withdrawal, which was attenuated by PKC inhibitors. plasma corticosterone levels were raised. SL-327, at doses that reduced the augmented pERK levels in the PVN, did not attenuate the rise in pCREB immunoreactivity or plasma corticosterone secretion. In contrast, PKC inhibition reduced the withdrawal-triggered rise in pCREB, pERK1/2 and corticosterone secretion. CONCLUSIONS AND IMPLICATIONS PKC mediated, in part, both CREB activation and the HPA response to morphine withdrawal. The ERK kinase/ERK pathway is probably not necessary for either activation of CREB or HPA axis hyperactivity. for 10 min at 4C. Samples comprising 40 g of protein were loaded on a 10% SDS/polyacrylamide gel, separated by electrophoresis and transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Non-specific binding of antibodies was prevented by incubating membranes in 1% bovine serum albumin in Tris-buffered saline Tween-20 (TBST; 10 mM Tris HCl, pH 7.6, 150 mM NaCl, Ro 31-8220 0.15% Tween-20). The blots were incubated with the following main antibodies: 1:750 polyclonal anti-phospho CREB-123-136 (pCREB; Millipore, Temecula, CA, USA); 1:1000 polyclonal anti-total CREB (Cell Signaling, Beverly, MA, USA); 1:1000 monoclonal anti-pERK1/2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); 1:6000 polyclonal anti-PKC (Sigma, St Louis, MO, USA); 1:1000 polyclonal anti-PKC (Santa Cruz); and 1:5000 polyclonal anti–tubulin (Cell Signaling), in TBST with bovine serum albumin over night at room heat (anti-phospho CREB, anti-total CREB and anti–tubulin) and 4C (anti-pERK1/2 and anti-PKC). After considerable washing with TBST, the membranes were incubated for 1 h at space heat with peroxidase-labelled secondary antibodies (anti-rabbit sc-2004 at 1:2500 for pCREB, 1:5000 for total CREB and 1:5000 for PKC and -tubulin; anti-mouse sc-2005 at 1:5000 for pERK1/2). After washing, immunoreactivity was recognized with an enhanced chemiluminescence’s Western blot detection system (ECL, Amersham Ro 31-8220 Ibrica, Madrid, Spain) and visualized by Amersham Hyper film-ECL. We used -tubulin (rabbit polyclonal antibody, Cell Signaling #4967) as our loading control for all the experiments. Before re-probing, blots were stripped by incubation with stripping buffer (glycine 25 mM and SDS 1%, pH 2) for 1 h at 37C. Blots were consequently reblocked and probed with anti–tubulin (1:5000, over night at room heat). Quantification of immunoreactivity related to pCREB (43 kDa), total CREB (43 kDa), ERK1 (42 kDa), ERK2 (44 kDa), PKC (80 kDa), PKC (80 Ro 31-8220 kDa) and -tubulin (52 kDa) bands was carried out by densitometry using Gel Doc (Bio-Rad, Hercules, CA, USA). The built-in optical density of the bands was corrected by subtraction of the background ideals. The ratios of pCREB/ -tubulin, total CREB/-tubulin, pERK1/ -tubulin, pERK2/ -tubulin, PKC/-tubulin and PKC/-tubulin were determined and indicated as a percentage of the average of settings in each blot. Immunohistochemical detection of CREB, p-CREB and CRF Sixty minutes after administration of saline or naloxone, rats were deeply anesthetized with an overdose of pentobarbital (100 mgkg?1) and perfused transcardially with PBS (pH 7.4) following by fixative containing paraformaldehyde (4% in PBS). After removal of the perfused brains, they were post-fixed in the same fixative and stored at 4C over night, Free-flotation serial coronal mind sections (30 m thickness) throughout the rostrocaudal extend of the hypothalamus were obtained on a freezing microtome Leica, Nussloch, Germany). Sections were pre-incubated in complete methanol comprising 10% H2O2, rinsed in PBS and treated with normal goat serum (NGS)CPBS (PBS comprising 2% swine serum and 0.5% Triton-X-100). Section were then incubated for 60 h at 4C with rabbit anti-total CREB (1:1500; Cell Signaling), rabbit anti-pCREB antibody (Upstate; 1:750 in NGSCPBS) or rabbit anti-CRF antibody (1:5000 in 2% NGSCPBS; a nice gift HPGD from Wylie Vale, The Salk Institute, La Jolla, CA, USA). This was followed by software of a biotinylated anti-rabbit IgG (diluted 1:200 for 1 h or 2 h) in NGSCPBS (Vector, Burlingame, CA), and then with the avidinCbiotin complex at space heat for 1 h. Visualization of the antigenCantibody reaction sites was performed using 0.033% 3,3-diaminobenzidine (DAB; Sigma, St Louis, MO, USA) and 0.014% H2O2 in 0.05 M TrisCHCl buffer. The reaction was halted in PBS. The sections were mounted on gelatine coated slides, dehydrated through graded alcohols, cleared in xylene and cover slipped with dibutylphthalate. Double-labelling immunohistochemistry of pCREB-immunoreactive nuclei and CRF-positive neurons For pCREB and CRF double-label immunohistochemistry, tissue sections from each rat in each treatment group were processed as follows: pCREB.
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