Determination of Gs and ADRmRNA by In Situ Hybridization

Sense and antisense oligonucleotide probes complementary to amino acids 379 to 394 of rat Gsa were 35S-labeled by the terminal transferase reaction. A partial rat i-ADR-cDNA was generated by PCR using primers annealing upstream of the third transmembrane domain and downstream of the fifth transmembrane domain spanning 506bp of the coding region (40). PCR fragments of the expected size were electroeluted after size selection, subcloned into the vector pBS SK- (Stratagene) and analyzed by terminal sequencing as described (62). 35S-labeled bl-ADR sense and antisense ribonucleotide probes were generated by in vitro transcription with T3 or T7 polymerase (Boehringer Mannheim) after plasmid linearization with Xhol or EcoRI, respectively. In situ hybridization of Gsa and prADR was performed according to published procedures (63,64,65,50,51).

2.3. Fura-2 Calcium Imaging of Rat and Mouse Pinealocytes

The intracellular concentration of free calcium ions ([Ca2+]i) of isolated rat and C3H pineal cells was analyzed with an Attofluor ratio imaging system (Zeiss, Germany; for details see 57). Changes in [Ca2+]i of single cells are expressed in a semiquantitative manner as 334 nm/380nm emission ratios. Analyzed cells were identified as pinealo-cytes by means of S-antigen immunoreaction (38,57).

2.4. Determination of pCREB by Immunocytochemistry

After one to three days in culture, isolated pinealocytes were stimulated as indicated, fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10min and then washed in PBS. To block endogenous peroxidase, the preparations were treated with methanol containing 0.45 % hydrogen peroxide for 10 min. After washing and preincubation with 10% normal goat serum (30min), the specimens were incubated with a polyclonal antibody against the serine 133-phosphorylated form of CREB (pCREB; 1 : 500). The primary antibody was dissolved in PBS containing 0.3% Triton X-100 and 1 % bovine serum albumin. Binding of the antibody was visualized using 1) biotin-conjugated anti-rabbit IgG as second antibody, 2) HRP-conjugated streptavidin as the third antibody, and 3) diaminobenzidine (DAB) as the coloring reagent. Some preparations were extensively washed and subsequently double-labeled with an S-antigen antibody (diluted 1 : 1000) to identify pCREB-immunoreactive cells as pinealo-cytes (37,38). Binding of the S-antigen antibody was visualized with a FITC-conjugated anti-rabbit IgG (Dako, Copenhagen, Denmark, diluted 1 : 200) (38,72).

2.5. Identification of Protein Kinases Involved in CREB Phosphorylation

Cultured organs or isolated pinealocytes were stimulated with NE with or without protein kinase inhibitors as indicated or left untreated as control. The PKA antagonist Rp-8-CPT-cAMPS was purchased from BioLog Lifescience Inst. (Bremen, Germany). The action mode of this class of antagonists has been described in detail (13,28,20). Chelerythrin and KN-62 were obtained from Calbiochem (Bad Soden, Germany), PD98059 from New England Biolabs (Schwalbach, Germany) and H-89 from BioMol (Hamburg, Germany).

2.6. Determination of PKA Subunit mRNA by Northern Blotting and

ReverseTranscriptase Polymerase Chain Reaction (RT-PCR)

To analyze expression of the PKA subunit transcripts, RNA extraction was performed according to a modification of a previously described procedure (42,16) or by use of the Qiagen Mini-RNA kit. A single pineal gland or 100,000 cells were extracted in 300^l Qiagen lysis buffer, processed according to the supplier's protocol and finally eluted into 50^l DEPC water. Northern blots of total RNA samples obtained from rat pineal glands six hours after light on (midday; 12:00) and six hours after light off (midnight; 24:00) were hybridized with cDNA probes against human RIa, RIP, RIIa and RII . The use of human cDNA probes for the detection of rat mRNA is possible since the interspecies homology for PKA subunit isoforms is more than 90% at the nucleotide level (26). Thus, human probes were expected to detect rat mRNA as has also been shown for bovine mRNAs (42). One to 3^l of the above described RNA preparations were used for a single reverse-transcriptase reaction performed with

4 MgCl2 (25mM), 2 ^l PCR buffer (l0x) (Perkin-Elmer, Konstanz, Germany), 8^l dNTP-mix (2.5mM each) (Roth, Germany), lpl oligo-dT primer (50^M), RNAse inhibitor (optional) and 1 ^l MuLV-reverse transcriptase (Perkin-Elmer, Konstanz, Germany). The reaction conditions were 25°C for l0min, 42°C for 15min, 99°C for

5 min and cooling down to 4°C until use or long term storage. Twenty ^l of the reversely transcribed DNA were directly diluted into 80^l PCR-mix and amplified. Primers and amplification protocols for the PKA regulatory subunits were used as described (71). The Taq-polymerase was from Bio Labs Ltd., Israel. The primers for GAPDH were 5 '-TGA-TGA-CAT-CAA-GAA-GGT-GG-3' (forward) and 5 '-TTT-CTT-ACT-CCT-TGG-AGG-CC-3' (reverse). All primers were purchased from MWG Biotech (Munich, Germany).

2.7. Determination of PKA Subunits by Immunoblotting

Cultured organs or isolated pinealocytes were stimulated with NE as indicated or left untreated as control. The medium was then removed and preparations were transferred into sample buffer. Electrophoresis and blotting was done as described (42). The membranes were incubated with polyclonal antibodies against the following sub-units of PKA: 1) catalytic subunit (C; 40 kDa; 1 : 50,000; 59), 2) regulatory subunit type Ia (RIa; 49kDa; 1 : 15,000; 11), 3) regulatory subunit type IP(RIP; 54 kDa; 1 : 1,000; 69), 4) regulatory subunit type IIa (RIIa; 54kDa; 1: 15,000; 11), 5) regulatory subunit type IIP (RIIP; 52kDa; 1 : 15,000; 69). The membranes were subsequently incubated with a horseradish peroxidase (HRP)- conjugated secondary antibody (New England BioLabs, Beverly, MA, USA) diluted 1 : 100,000. The signals were obtained by chemi-luminescence detection on autoradiographic film (Ultrasignal reagent and Clxposure film, Pierce, Rockford, II., USA). To confirm equal loading of the lanes, immunoblots were stained with India Ink (Pelikan, Hannover, Germany) after chemiluminescence detection. Both, autoradiograms and stained blots were analyzed densitometrically (see below).

2.8. Detection of PKA Catalytic Subunit Translocation

Pinealocytes were treated for 1 hour with NE or left untreated. Nuclear extracts were prepared as previously described (23). In brief, pineal preparations were harvested in AT buffer (60mM KCI, 15mM NaCl, 14mM P-mercaptoethanol, 2mM EDTA, 15 mM HEPES pH 7.9, 0.3 M sucrose, 5 ng/ml aprotinin, 10 ng/ml leupeptin, 2^g/ml pepstatin, 0.1 mM phenylmethylsulfonyl-chloride, 1 mM NaF, 1 mM N3VO3 and 1 mM Na3MnO4) containing 0.1 % Triton X-100 and homogenized.The homogenate was layered onto AT-buffer containing 1 M sucrose and centrifuged for 5 min at 8.000 g in an Eppendorf centrifuge to collect the nuclear fraction in the pellet. The supernatant contains the cytosolic fraction. The protein content of each fraction was measured and adjusted to 1 ^g/^l with 2x electrophoresis sample buffer.

Visualization of the PKA catalytic subunit (1 : 1000) was done by immuno-fluorescence (see above) using a CY3-conjugated anti-rabbit secondary antibody (1 : 1000; Dianova, Hamburg, Germany). Confocal images were obtained using the software package of a LSM510 connected to an Axi overt 100 microscope (Zeiss, Jena, Germany).

2.9. Computer-Assisted Semiquantitative Analysis of PKA-Subunit

Immunoblots and pCREB Immunocyto-Chemistry

Computer-based image analysis systems (VIDAS and KS 300 systems, Kontron, Eching, FRG) were used to carry out a combined plani- and densitometric semiquantitative analysis of the data of the PKA subunit immunoreaction from immunoblots and the pCREB signals from immunocytochemical preparations (72; Wicht et al., in press). The immunoblots were digitized with a scanner (constant settings for all measurements: grey value resolution 8 bits, spatial resolution 200 pixel per inch, fixed settings of the A/D-converter). In order to achieve higher numerical values for darker (more intense) signals, the images were inverted (light areas were represented by low grey-values, dark areas by high values). The immunoreactive signals were segmented from the background; their size and intensity were quantified simultaneously by summing up the grey values of all pixels belonging to an individual signal. The results are given as "SUMDENS" (sumdensity) values. A plot of the amount of antigen against the SUMDENS values shows a linear relation between the SUMDENS values and the logarithm of the antigen concentration (Wicht et al., unpublished observation). A very similar method was used to quantify the immunocytochemical results of the pCREB reaction in dispersed pinealocytes. A light microscope equipped with a constant light source and black/white CCD-camera was used to record digital images from various regions in these preparations (constant settings for all recordings: 20x-objective , grey value resolution 8 bits, spatial resolution 512 x 512 pixel/image, fixed settings of the A/D-converter of the CCD-camera). Typically, four regions per culture were analyzed and care was taken to select only regions that contained an equally and comparably dense monolayer of cells. The unstained background between the cells was used as an intensity reference and was adjusted to a grey value of 255 by slight variations of the intensity of the light source. The resulting digital images required only minimal image processing and allowed the discrimination and segmentation of the immunoreactive nuclei using the same discrimination value for all measurements. After the segmentation, the sum of all (inverted, see above) grey values of the pixels belonging to the immunoreactive nuclei was determined; the results are also given as "SUMDENS" values. The SUMDENS values need to be corrected for the total area covered by cells. The resulting parameter is called SUMDENSCORR.

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