Introduction

Top Abstract Introduction Experimental Procedures Results Discussion References

Several clinical and preclinical studies suggest that adrenal steroids are involved in various functional aspects of the central nervous system such as behavior, emotion, and learning (for review, see McEwen, 1987). This is evident from the studies suggesting that abnormal hypothalamic-pituitary-adrenal (HPA) functions are associated with affective disorders. For example, higher levels of cortisol in plasma and of corticotrophin-releasing hormone in cerebrospinal fluid, and failure to suppress plasma cortisol levels after administration of dexamethasone (also known as the dexamethasone suppression test) have been shown in a subgroup of depressed patients (for review, see Nelson and Davis, 1997). In addition, glucocorticoid treatment induces depression (Ling et al., 1981), and antiglucocorticoids are effective antidepressants (for review, see Wolkowitz and Reus, 1999). Also, Fernandes et al. (1997) recently showed that protracted treatment with corticosterone (CORT) induces behavioral depression in rats.

The precise mechanisms by which behavioral changes occur because of abnormal HPA functions are not clear; however, adrenal steroids have been shown to alter the expression of many neurotransmitter receptors, including 5-hydroxytryptamine (5HT)_1A, 5HT_2A, 5HT_2C, -aminobutyric acid, and -adrenergic receptors (Meyer, 1985; Kuroda et al., 1993; Chaouloff, 1995). Besides their genomic actions, emerging evidence suggests that adrenal steroids may use receptor-mediated signaling pathways to regulate a wide range of functions over a broader time scale (for review, see Moore and Evans, 1999). In this regard, we examined the possible involvement of the phosphatidylinositol (PI) signaling pathway, which has been shown to be linked with many receptors, including 5HT_2A, 5HT_2C, and ₁-adrenergic receptors, in glucocorticoid-mediated action.

In the PI signaling pathway, agonist-induced interaction of cell surface receptors with guanine nucleotide binding proteins (G proteins) activates the enzyme PI-specific phospholipase C (PI-PLC), which catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG) (Berridge and Irvine, 1989). Both IP₃ and DAG act as second messengers. Whereas IP₃ mobilizes Ca²⁺ from intracellular sources after binding with IP₃ receptors (Berridge et al., 1998), DAG activates the phosphorylating enzyme protein kinase C (Dekker et al., 1995). These events mediate cellular activation and subsequent biological responses such as neurotransmitter release, cell growth, differentiation, neuronal development, and gene expression (Nishizuka, 1988; Berridge and Irvine, 1989). Our earlier studies suggested that chronic but not acute administration of dexamethasone, a synthetic glucocorticoid, increases the catalytic activity of PI-PLC and the mRNA and protein expression of a specific isozyme, PLC ₁, in rat brain (Dwivedi and Pandey, 1999a). Also, it was previously reported that in vitro addition of dexamethasone to C6 glioma cells mobilizes 5HT_2A receptor-mediated Ca²⁺ (Muraoka et al., 1993) and that chronic administration of dexamethasone to rats causes decreased norepinephrine-stimulated PI metabolism in the brain (Takahashi et al., 1996). Because administration of dexamethasone inhibits the release of CORT via a feedback mechanism and is a poor substitute for depleted endogenous glucocorticoids in the brain (Birmingham et al., 1993), from these studies it is not clear whether the effects produced by dexamethasone are in response to endogenous glucocorticoids and whether the administration of exogenous glucocorticoids has similar or opposite effects to those elicited by dexamethasone.

To clarify these issues, in the present investigation, we studied the effects of both endogenous and exogenous glucocorticoids on PI-PLC activity and on the expression of PLC isozymes in various brain areas by adrenalectomizing (ADX) rats and simultaneously implanting CORT pellets into ADX rats, as well as by implanting CORT pellets of various doses into normal rats. Because G_q  proteins are coupled to and activate the enzyme PI-PLC (Strathmann and Simon, 1990), we studied their expression in brain of these rats. Furthermore, given the significance of IP₃ in mobilizing Ca²⁺ from intracellular sources and mediating the signaling further downstream, we examined the effects of exogenous and endogenous glucocorticoids on levels of IP₃, binding of [³H]IP₃ to IP₃ receptors (IP₃Rs), and on the expression of various isoforms of IP₃Rs in brain of these rats. This investigation not only clarifies whether changes in PI-PLC are associated with endogenous glucocorticoids but also helps elucidate the role of IP₃ in glucocorticoid-mediated actions and, ultimately, the involvement of these components of the PI signaling system in HPA axis-mediated changes in mood and behavior.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

Materials

D-IP₃, phosphatidylinositol 4,5-biphosphate (PIP₂), hexadecyltrimethylammonium bromide (cetrimide), and monoclonal antibody for -actin were purchased from Sigma Chemical Co. (St. Louis, MO). Monoclonal antibodies for PLC ₁, ₁, and ₁ were obtained from Upstate Biotechnology (Lake Placid, NY). Antibodies for IP₃R-I, IP₃R-II, and IP₃R-III were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). [³H]PIP₂, Hot Tub DNA polymerase, RNase inhibitor, BglII, [-³²P]dCTP, horseradish peroxidase-linked secondary antibodies (anti-mouse/rabbit), enhanced chemiluminiscence (ECL)-nitrocellulose membrane, chemiluminescent detection reagent, and the IP₃ assay kit were obtained from Amersham (Arlington Heights, IL). EcoRI, HindIII, and the in vitro transcription kit were purchased from Promega (Madison, WI). CORT pellets were from Innovative Research of America (Sarasota, FL).

Animals

Virus-free Sprague-Dawley male rats initially weighting 220 to 220 g were used. Rats were housed in groups of three under standard laboratory conditions (temperature 21 ± 1°C, humidity 55 ± 5%, 12-h light/dark cycle). Animals were provided free access to food. Rats were acclimatized for 1 week before the experiment started.

ADX and Treatment with CORT

Two treatment protocols were used. In the first protocol, rats under light halothane anesthesia were s.c. implanted with CORT pellets containing 50 or 100 mg of CORT in a cholesterol base. These CORT pellets can maintain a physiological serum concentration of CORT for 21 days. The release of CORT after implantation of a 50- or 100-mg CORT pellet is 2.38 or 4.76 mg/day, respectively. Control rats underwent an identical surgery procedure with implantation of a cholesterol base pellet or underwent no treatment; these two types of treatment did not differ in their results in the final determinations of PI-PLC activity, of [³H]IP₃ receptor binding, or of protein levels of PLC isozymes or IP₃R isoforms. Rats were decapitated 1 or 14 days after pellet implantation. In the second protocol, rats were anesthetized with halothane anesthesia. Bilateral ADX was performed by making a small incision (0.5 in.) in the skin and the muscle wall just below the ribcage. The adrenal glands were visualized and removed. The muscle wall was sutured, and the skin incision was closed with wound clips. Control rats were sham-operated; the adrenal glands were visualized but not removed. These rats were given drinking water containing 0.9% (w/v) saline ad libitum. They were decapitated on day 1 or 14 after ADX. Some ADX rats were implanted s.c. with placebo or a CORT pellet (containing 50 or 100 mg of CORT) immediately after ADX. The rats were decapitated 1 or 14 days after CORT pellet implantation.

The trunk blood was collected on ice at decapitation and was centrifuged, and then the serum was stored at 80°C until the assays were performed. Serum CORT levels were measured by a commercially available radioimmunoassay kit (ICN Biomedical, Inc., Cleveland, OH). Brains were removed quickly after the blood was taken. Cortex, hippocampus, and cerebellum were dissected out and immediately stored at 80°C until analyzed. For both experimental protocols, rats were decapitated between 9:00 AM and 11:00 AM, corresponding to 3 to 5 h after lights on.

Preparation of Membrane and Cytosol Fractions

Cortices and hippocampi were homogenized in a homogenizing buffer containing 20 mM Tris-HCl (pH 7.4), 2 mM EGTA, 5 mM EDTA, 1.5 mM pepstatin, 2 mM leupeptin, 0.5 mM phenylmethylsulfonyl fluoride, 0.2 U/ml aprotinin, and 2 mM dithiothreitol, using the Polytron. The supernatant was centrifuged at 100,000g for 60 min at 4°C. The resulting supernatant was the cytosol fraction, and the pellet was resuspended in the homogenizing buffer containing 0.2% (w/v) Triton X-100. The homogenate was kept at 4°C for 60 min with occasional stirring and then centrifuged at 100,000g for 60 min at 4°C. The resulting supernatant was used as the membrane fraction. Both the membrane and the cytosol fractions were used to determine the PI-PLC activity and the immunolabeling of PLC isozymes. The concentration of protein in these fractions was determined using the procedure of Lowry et al. (1951).

Determination of PI-PLC Activity

PI-PLC activity was measured in membrane and cytosol fractions of cortex and hippocampus by the enzymatic assay procedures described by Dwivedi and Pandey (1999a) using 5 µg of protein/tube in an incubation buffer (20 mM Tris-HCl, 1 mM CaCl₂, and 100 mM KCl, pH = 7.4) containing 10 mM lithium chloride, PIP₂ substrate (50 µM unlabeled PIP₂, 2.0 µCi/ml [³H]PIP₂, and 0.5 mg/ml cetrimide) in a total volume of 100 µl at 37°C for 10 min. The reaction was terminated by the addition of 500 µl of 1 M HCl and 500 µl of a mixture of chloroform:methanol (1:1 v/v). The tubes were vigorously mixed and centrifuged at 1000g for 10 min. The aqueous (upper) phase was transferred to a scintillation vial containing scintillation liquid, and the radioactivity was counted in a liquid scintillation counter. Each experiment had its blank, in which the protein suspension was added after stopping the reaction with chloroform:methanol. PI-PLC activity is expressed as the amount of [³H]IP₃ formed (dpm) per minute per milligram of protein.

Immunolabeling of PLC ₁, ₁, and ₁ Isozymes

Equal volumes of protein samples (membrane or cytosol fractions; 20 µl containing 30 µg of protein) and gel loading solution [50 mM Tris-HCl (pH = 6.8), 4% 2-mercaptoethanol, 1% SDS, 40% glycerol, and a trace amount of bromphenol blue] were mixed and the samples were boiled for 3 min and kept on ice for 10 min. The samples (30 µg of protein in each lane) were loaded onto 7.5% (w/v) polyacrylamide gel using the Mini Protein II gel apparatus (Bio-Rad, Hercules, CA). The gels were run using 25 mM Tris base, 192 mM glycine, and 0.1% (w/v) SDS at 150 V. The proteins were subsequently transferred electrophoretically to an ECL-nitrocellulose membrane (Amersham) using the Mini Trans Blot transfer unit (Bio-Rad) at 0.150-amp constant current. Membranes were washed with Tris-buffered saline/Tween 20 (TBST) buffer (10 mM Tris base, 0.15 M NaCl, and 0.05% Tween 20) for 10 min. The blots were blocked by incubating with 5% (w/v) powdered nonfat milk in TBST, 0.2% (v/v) Nonidet P-40, and 0.02% (w/v) SDS (pH = 8.0). Then the blots were incubated for 1 h with primary monoclonal antibody (anti-PLC ₁, ₁, or ₁) at a dilution of 1:1000 (Dwivedi and Pandey, 1999a). The membranes were then washed with TBST and incubated with horseradish peroxidase-linked secondary antibody (anti-mouse IgG; 1:3000) for 1 h at room temperature. The membranes were then extensively washed with TBST and exposed to ECL film. Before starting the immunolabeling experiments with the samples, the procedure was standardized using 10 to 200 µg of protein. We found that the optical density of the bands varied linearly with concentration up to 100 µg of protein. To normalize our data, we measured -actin in the same immunoblot using -actin as the monoclonal primary antibody (1:5000 for 2 h) and anti-mouse IgG (1:5000 for 2 h) as the secondary antibody. In addition, the dilution of the antibodies and the duration of exposure of the nitrocellulose membranes on autoradiographic film were standardized. The optical densities of the bands on the autoradiograms were quantified using the Loats Image Analysis System (Westminster, MD), and the optical density of each PLC isozyme band was corrected by the optical density of the corresponding -actin band. The values are represented as a percentage of the control values.

Determination of mRNA Levels of the PLC ₁ Isozyme by Competitive Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

The procedure for determining the mRNA levels of the PLC ₁ isozyme in rat brain has been described in detail by Dwivedi and Pandey (1999a).

RNA Isolation. Cortices and hippocampi were homogenized in 4 M guanidine isothiocyanate, 50 mM Tris-HCl (pH = 7.4), and 25 mM EDTA, and the total RNA was isolated by CsCl₂ ultracentrifugation. The yield of total RNA was determined by measuring the absorbency of an aliquot of the precipitated stock at a wavelength of 260/280 nm. To check for possible DNA contamination, after each extraction, samples were run by RT-PCR without adding the reverse transcriptase enzyme.

Oligonucleotides. Amplification primers were synthesized on the model 381A DNA synthesizer (Applied Biosystems, Foster City, CA) by using phosphoramidite chemistry, leaving the terminal dimethoxytrityl group intact. All primers were purified by reverse-phase chromatography using oligonucleotide purification columns (Applied Biosystems) according to the manufacturer's manual. The primer pairs were designed to allow amplification of 532 to 847 base pairs (bp): forward, 5'-TTTTCGGCAGACCGGAAGCGA-3', and reverse, 5'-TGCTGTTGGGCTCGTACTTCT-3' (Suh et al., 1988; GenBank accession no. M20636). Each primer contained a comparable G/C content to minimize variability in hybridization efficiency at the annealing temperature. The specificity of the PLC ₁ mRNA product was checked by sequencing the amplified area with the Sequenase version 2.0 DNA sequencing kit using HindIII and EcoRI, which produced fragments of the expected size.

Synthesis and Cloning of Internal Standards. The detailed procedures for the preparation of internal standards (cRNA) have been described by Dwivedi and Pandey (1999a). Each internal standard targeted by the same primers used to amplify the canonic sequence was generated by site-directed mutagenesis to introduce a BglII restriction site between the amplification primers so that the digestion of the amplicon would generate two fragments of approximately equal molecular size. The internal primer sequence was as follows: (645-668 bp) 5'-CCTGAA AGATCTTCCCCGACC-3'. The underlined bases indicate the BglII restriction site, whereas bold and italicized bases indicate the mutation sites. The single-strand internal primers were designed and synthesized so that the restriction site was introduced with only a minimal number of base substitutions, and also such that there was a 21-bp overlap of the primary PCR products. Each of the internal standards was synthesized in two PCR steps, starting with a cDNA template reverse-transcribed from the total RNA. The internal standard template was linearized with SspI. The cRNA corresponding to sense strand was synthesized with linearized template and Sp6 RNA polymerase by means of an in vitro transcription kit.

Quantitative Analyses of PLC ₁ Isozyme mRNA by Competitive RT-PCR. Decreasing concentrations of PLC ₁ internal standard cRNA were added to 1 µg of total RNA isolated from cortices or hippocampi. The RNA/cRNA mixtures were denatured at 80°C for 6 min and then reverse-transcribed with cloned Moloney murine leukemia virus and reverse transcriptase (200 U; Life Technologies, Grand Island, NY) in RT buffer containing 50 mM Tris-HCl (pH = 8.3), 75 mM KCl, 3 mM MgCl₂, and 1 mM dNTPs using random hexamers (2.5 mM) and human placenta ribonuclease inhibitor (HPRI) (8.3 U) in a volume of 20 µl. The RT mixture was incubated at 37°C for 60 min to promote cDNA synthesis. The reaction was terminated by heating the samples at 98°C for 5 min. In all assays, as a control, one RT reaction was performed in the absence of RNA.

Competitive PCR Amplification. After termination of the RT reaction, cDNA aliquots containing reverse-transcribed material were amplified with Hot Tub DNA polymerase in the Thermal cycler (9600; Perkin-Elmer, Norwalk, CT). The amplification mixture contained cDNA, 0.5 µM specific primer pairs, 1.5 mM MgCl₂, 50 mM Tris-HCl (pH = 9.0), 20 mM ammonium sulfate, 15 mM KCl, and 0.5 U of Hot Tub DNA polymerase in a 100-µl volume. Trace amounts of [³²P]dCTP (0.5-1 µCi/sample) were included during the PCR step for subsequent quantification. The PCR mixture was amplified for 32 cycles with denaturation (94°C; 15 s), annealing (60°C; 30 s), and elongation (72°C; 30 s) amplification steps. The reaction was terminated with a 5-min final elongation step. After amplification, aliquots were digested with BglII in triplicate and run by 1.5% agarose gel electrophoresis.

Preparation of Membranes for [³H]IP₃ Binding Assay and Immunolabeling of IP₃R Isoforms

Brain samples were homogenized by Polytron at setting 7 for 30 s in a homogenizing buffer containing 50 mM Tris-HCl (pH = 7.7), 1 mM EDTA, and 2 mM 2-mercaptoethanol. The homogenate was centrifuged at 40,000g for 15 min at 4°C. The supernatant was discarded and the pellet was homogenized once again in the homogenizing buffer and centrifuged as described above. This process was repeated one more time and the resulting pellet was resuspended in a buffer containing 50 mM Tris-HCI (pH = 8.4), 1 mM EDTA, and 1 mM 2-mercaptoethanol. This fraction was used for the [³H]IP₃ binding assay, as well as for the immunolabeling of IP₃Rs.

[³H]IP₃ Binding Assay

Binding of [³H]IP₃ was carried out in duplicate by the procedures described in Dwivedi et al. (1998). The incubation medium contained incubation buffer (50 mM Tris-HCI, pH = 8.4, 1 mM 2-mercaptoethanol, 1 mM EDTA), [³H]IP₃ (specific activity 21 Ci/mmol) ranging from 10 to 100 nM (six different concentrations), and 40 µl of membrane suspension in a total volume of 100 µl. Nonspecific binding was determined in the presence of 10 µM D-IP₃. The incubation was performed at 4°C for 10 min and rapidly terminated by the addition of 5 ml of cold washing buffer [50 mM Tris-HCI, pH = 7.7, 1 mM EDTA, and 0.1% (w/v) BSA] and filtration under vacuum through Whatman GF/B filters. The filters were dried and the filter-bound radioactivity was analyzed by a liquid scintillation counter. Specific binding was defined as the difference between total binding and binding observed in the presence of D-IP₃. The maximum number of binding sites (B_max) and the apparent dissociation constant (K_D) were computed by Scatchard Analysis using the EBDA program (McPherson, 1985). The protein concentration in membrane fraction was determined by the method of Lowry et al. (1951).

Immunolabeling of IP₃R Isoforms

The immunodetectable levels of IP₃R-I, IP₃R-II, and IP₃R-III isoforms were determined by Western blot as described above for PLC isozymes and also in our previous publication (Dwivedi et al., 1998). Equal amounts of protein samples (25 µg) were subjected to a 7.5% polyacrylamide gel and subsequently transferred to an ECL- nitrocellulose membrane. The blots were incubated with primary anti-IP₃R-I, -IP₃R-II, or -IP₃R-III (1:2,000 for overnight at 4°C) or -actin (1:5,000 for 2 h) and secondary (anti-goat/mouse) antibodies (1:2,500-1:10,000 for 3-5 h, depending on the primary antibody used).

Determination of IP₃ Levels

Brain samples were processed for determining IP₃ levels by homogenization in ice-cold buffer (0.4 M HClO₄ containing 1 mM EDTA). The homogenate was centrifuged at 30,000g for 15 min at 4°C (Meek, 1986). The resulting supernatant was titrated to pH = 7.5 with 1.53 M KOH containing 75 mM HEPES and kept on ice for 60 min. The solution was centrifuged at 2000g for 15 min at 4°C and aliquots of the supernatant were used for determining IP₃ levels using an IP₃ assay kit (Amersham). Briefly, the assay procedure is as follows: 100 µl of protein samples (containing 10 µg of protein) was added to 100 µl of assay buffer (0.1 M Tris, pH = 9.0, 4 mM EDTA, 4 mg/ml BSA), 100 µl [³H]IP₃ (6.8 nCi), and 100 µl of bovine adrenal binding protein. In a separate experiment nonspecific binding was determined by adding 100 µl of D-myo IP₃ standard (400 pmol) instead of protein samples, and zero standard was determined by removing the protein sample and the standard from the assay medium. A standard curve was plotted by using D-myo IP₃ standard (0.19-25 pmol, eight different concentrations), to which no protein samples were added. The reaction was carried out for 15 min on ice. Tubes were then centrifuged at 1000g for 3 min at 4°C. The supernatant was discarded and the pellet was suspended in 200 µl of water. Two milliliters of scintillation cocktail was added to each tube, and after mixing, the radioactivity was counted in a beta counter. IP₃ levels were determined by interpolation from a standard curve. The results are expressed as picomoles of IP₃ per milligram of protein.

Immunolabeling of G_q/11  Protein

Gel electrophoresis and immunolabeling of G_q/11  protein were performed by the Western blot technique using a method described by Dwivedi and Pandey (1997). The tissues were homogenized in a buffer containing 50 mM Tris-HCl (pH = 7.5), 1 mM EDTA, 2 mM MgCl₂, 1 mM phenylmethylsulfonyl fluoride, 5 mM iodoacetamide, and 0.2 U/ml aprotinin and centrifuged at 3000 rpm for 10 min. The supernatant was recentrifuged at 32,000 rpm for 15 min, and the resultant pellet was resuspended in the same buffer. Protein content in the samples was determined by the method of Lowry et al. (1951) using BSA as a standard. Aliquots (15 µl) of membrane suspension were added to 15 µl of 50 mM Tris-HCl (pH = 6.8), 1 mM dithiothreitol, and 2% SDS and incubated for 5 min at 75°C. After incubation, 20 µl of 100 mM N-ethylmaleimide was added, and samples were incubated for another 15 min at 21°C. Finally, 50 µl of gel loading solution was added and the samples were boiled for 3 min and kept on ice for 10 min. The samples (30 µg of protein in each lane) were loaded onto a 10% (w/v) acrylamide gel. Gel electrophoresis and subsequent steps were performed similarly as described previously for the immunolabeling of PLC isozymes. The blots were incubated overnight at 4°C with primary monoclonal antibody (anti-G_q/11 ) at a dilution of 1:3000 and with horseradish peroxidase-linked secondary antibody (anti-rabbit IgG, 1:3000) for 3 h at room temperature. The filters were extensively washed with TBST and exposed to ECL film. -Actin was run in the same blot. The bands on the autoradiograms were quantified as described previously for the immunolabeling of PLC isozymes.

Statistics

Data were analyzed using the SPSS 9.0 (Chicago, IL) statistical software package. All values are given as the mean ± S.D. Data were subjected to one-way ANOVA followed by Dunnett's test for multiple comparisons (Dunnett, 1980). An -value lower than .05 was considered significant.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Serum CORT Levels. The serum CORT levels 1 day after ADX (acute) were as follows: sham, 52.5 ± 14.1; ADX, 0.3 ± 0.2 ng/ml, whereas at day 14 (chronic), we could not detect CORT in ADX rats. The levels of CORT in serum 14 days after CORT pellet implantation into ADX rats were as follows: ADX + CORT (50-mg pellet), 25.2 ± 6.7; ADX + CORT (100-mg pellet), 47.8 ± 15.1 ng/ml. CORT pellet implantation into normal rats yielded the following serum CORT levels at day 14: CORT (50-mg pellet), 60.1 ± 17.5; CORT (100-mg pellet), 110 ± 19.5 ng/ml. This is in agreement with our previous observations (Dwivedi and Pandey, 2000)

Effects of ADX and of Simultaneous CORT Pellet Implantation into ADX Rats on PI-PLC Activity. It was observed that 1 day after ADX there were no significant effects on PI-PLC activity either in cortex (membrane: sham, 20,659 ± 1,967; ADX, 21,137 ± 2,034 dpm/min/mg of protein; cytosol: sham, 22,128 ± 2,153; ADX, 21,865 ± 1,553 dpm/min/mg of protein) or in hippocampus (membrane: sham, 29,511 ± 2,175; ADX, 31,165 ± 2,017 dpm/min/mg of protein; cytosol: sham, 33,511 ± 3,014; ADX, 31,197 ± 1,668 dpm/min/mg of protein).

View larger version (46K): [in this window] [in a new window]   Fig. 1.   Effects of 14 days of ADX and of ADX + CORT pellet implantation (50 or 100 mg) on PI-PLC activity in membrane and cytosol fractions of cortex (A) and hippocampus (B). , sham-operated; , ADX; , ADX + CORT (50 mg); , ADX + CORT (100 mg). CORT pellet (50 or 100 mg) was implanted immediately after ADX. Values are the mean ± S.D. from six rats in each group. The ADX group was compared with the sham-operated group, whereas the ADX + CORT-treated groups were compared with the ADX group. #, as compared with sham; ##, as compared with ADX. *P < .01, **P < .001.

Effects of CORT Treatment of Normal Rats on PI-PLC Activity. Acute (1-day) implantation of a CORT pellet into normal rats, either 50 or 100 mg, had no significant effects on PI-PLC activity in membrane or cytosol fractions of cortex or hippocampus (data not shown). However, as shown in Fig. 2, at 14 days both doses of CORT had significantly decreased the catalytic activity of PI-PLC, the higher dose (100 mg) being more effective in both membrane and cytosol fractions of cortex (Fig. 2A) and hippocampus (Fig. 2B) than the lower dose (50 mg).

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Effects of 14 days of CORT pellet implantation (50 or 100 mg) into normal rats on PI-PLC activity in membrane and cytosol fractions of cortex (A) and hippocampus (B). , sham-operated; , CORT (50 mg); , CORT (100 mg).Values are the mean ± S.D. from six rats in each group. CORT-treated groups were compared with the sham-operated group. *P < .001.

Effects of ADX and of Simultaneous CORT Pellet Implantation into ADX Rats on Immunolabeling of PLC Isozymes (PLC ₁, ₁, and ₁) as Well as on mRNA Levels of the PLC ₁ Isozyme. We determined the immunolabeling of the various PLC isozymes (PLC ₁, ₁, and ₁) in both membrane and cytosol fractions of cortex and hippocampus. As reported in Dwivedi and Pandey (1999a), it was observed that PLC ₁ migrated to 150 kDa, whereas PLC ₁ and ₁ migrated to 85 and 145 kDa, respectively (Fig. 3). The molecular mass of -actin, used as a housekeeping protein, was 46 kDa (Fig. 3). The immunolabeling of -actin in cortex and hippocampus did not change either after ADX or after CORT pellet implantation into normal rats. This is in agreement with our previous reports suggesting that levels of -actin remain unaltered in rat brain after treatment with dexamethasone or CORT (Dwivedi and Pandey, 1999a,b, 2000).

View larger version (56K): [in this window] [in a new window]   Fig. 3.   Representative Western blots of PLC isozymes in membrane and cytosol fractions of cortex 14 days after ADX and after implantation of CORT pellets (50 or 100 mg) into ADX rats.1, sham-operated; 2, ADX; 3, ADX + CORT (50 mg); 4, ADX + CORT (100 mg). Protein samples (30 µg) were electrophoresed on a 7.5% polyacrylamide gel, followed by electrotransfer to a nitrocellulose membrane. The blots were probed with monoclonal anti-PLC 1, 1, or 1 antibodies (1:1000 dilution). The molecular masses for the PLC 1, 1, and 1 isozymes were 150, 85, and 145 kDa, respectively. The blots were stripped and reprobed with -actin antibody (1:5000 dilution). -Actin migrated to 46 kDa. Ratios of the optical densities of PLC 1, 1, or 1 to the optical density of -actin were calculated.

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Effects of 14 days of ADX and of ADX + CORT pellet implantation (50 or 100 mg) on protein levels of PLC isozymes in membrane and cytosol fractions of cortex (A) and hippocampus (B). , sham-operated; , ADX; , ADX + CORT (50 mg); , ADX + CORT (100 mg). Values are the mean ± S.D. from six rats in each group. The ADX group was compared with the sham-operated group, whereas the ADX + CORT-treated groups were compared with the ADX group. #, as compared with sham; ##, as compared with ADX. *P < .01, **P < .001. Note that we did not observe any significant effects of ADX on protein levels of PLC 1 or 1 isozyme in cortex or hippocampus.

View larger version (56K): [in this window] [in a new window]   Fig. 5.   A, representative experiment showing a competitive PCR analysis for PLC 1 mRNA content in the cortex of a normal rat brain. Decreasing concentrations of PLC 1 standard cRNA (400-25 pg) were added to a constant amount (1 µg) of total RNA isolated from the hippocampus. The mixtures were reverse-transcribed and PCR-amplified in the presence of trace amounts of [32P]dCPT; aliquots were digested by BgIII and electrophoresed on 1.5% agarose gel. The higher molecular size band (316 bp) corresponds to the amplification products arising from the mRNA, whereas the lower band (214 + 122 bp) arises from cRNA generated from the internal standard digested by BgIII. B, data derived from the agarose gel are plotted as the counts incorporated into the amplified cRNA standard divided by the counts incorporated into the corresponding isozyme mRNA amplification product versus the known amount of internal standard cRNA added to the test sample. The point of equivalence represents the amount of PLC 1 mRNA.

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Effects of 14 days of ADX and of ADX + CORT pellet implantation (50 or 100 mg) on mRNA levels of the PLC 1 isozyme in cortex and hippocampus. , sham-operated; , ADX; , ADX + CORT (50 mg); , ADX + CORT (100 mg). Values are the mean ± S.D. from six rats in each group. The ADX group was compared with the sham-operated group, whereas the ADX + CORT-treated groups were compared with the ADX group. #, as compared with sham; ##, as compared with ADX. *P < .01, **P < .001.

Effects of CORT Treatment of Normal Rats on Immunolabeling of PLC Isozymes (PLC ₁, ₁, and ₁) and on mRNA Levels of the PLC ₁ Isozyme. Acute (1 day) implantation of a CORT pellet into normal rats, either 50 or 100 mg, failed to cause any changes in the immunolabeling of PLC ₁, ₁, or ₁ isozyme in cortex or hippocampus (data not shown).

View larger version (51K): [in this window] [in a new window]   Fig. 7.   Representative Western blots of PLC isozymes in membrane and cytosol fractions of cortex 14 days after implantation of CORT pellets (50 or 100 mg) into normal rats. 1, sham-operated; 2, CORT (50 mg); 3, CORT (100 mg). Protein samples (30 µg) were electrophoresed on a 7.5% polyacrylamide gel, followed by electrotransfer to a nitrocellulose membrane. The blots were probed with monoclonal anti-PLC 1, 1, or 1 antibodies (1:1000 dilution for 1 h). The molecular masses for the PLC 1, 1, and 1 isozymes were 150, 85, and 145 kDa, respectively. The blots were stripped and reprobed with -actin antibody (1:5000 dilution for 2 h). -Actin migrated to 46 kDa. Ratios of the optical densities of PLC 1, 1, or 1 to the optical density of -actin were calculated.

View larger version (17K): [in this window] [in a new window]   Fig. 8.   Effects of 14 days of CORT pellet implantation (50 or 100 mg) on protein levels of PLC isozymes in cortex and hippocampus. , sham-operated; , CORT (50 mg); , CORT (100 mg). Values are the mean ± S.D. from six rats in each group. CORT-treated groups were compared with the sham-operated group. *P < .001, **P < .001. Note that we did not observe any significant effects of 14 days of CORT treatment on the immunolabeling of PLC 1 or 1 isozyme in cortex or hippocampus.

View larger version (18K): [in this window] [in a new window]   Fig. 9.   Effects of 14 days of CORT pellet implantation (50 or 100 mg) into normal rats on mRNA levels of the PLC 1 isozyme in cortex and hippocampus. , sham-operated; , CORT (50 mg); , CORT (100 mg). Values are the mean ± S.D. from six rats in each group. CORT-treated groups were compared with the sham-operated group. *P < .001.

Effects of ADX and of Simultaneous CORT Pellet Implantation into ADX Rats on [³H]IP₃ Binding and on Immunolabeling of IP₃Rs. In our initial experiments, we characterized [³H]IP₃ binding in various areas of rat brain and found that B_max of [³H]IP₃ binding was highest in cerebellum, whereas in cortex and hippocampus, B_max was much lower. However, the affinity of [³H]IP₃ binding was higher in cortex and hippocampus than in cerebellum, which is in agreement with reports in the literature (Snyder and Supattapone, 1989); therefore, besides cortex and hippocampus, we also determined [³H]IP₃ binding in the cerebellum.

View larger version (66K): [in this window] [in a new window]   Fig. 10.   Representative Western blots showing the effects of 14 days of ADX and of ADX + CORT pellet implantation (50 or 100 mg) (A) or CORT pellet implantation (50 or 100 mg) into normal rats on the protein levels of IP3Rs in rat cortex (B). 1, sham-operated; 2, ADX; 3, ADX + CORT (50 mg); 4, ADX + CORT (100 mg); 5, sham-operated; 6, CORT (50 mg); 7, CORT (100 mg). Protein samples (25 µg) were electrophoresed on a 7.5% polyacrylamide gel, followed by electrotransfer to a nitrocellulose membrane. The blots were probed with anti-IP3R-I, -IP3R-II, or -IP3R-III antibody (1:2000 dilution). The apparent molecular masses of IP3R-I, IP3R-II, and IP3R-III were similar, ~300 kDa. The blots were stripped and reprobed with -actin antibody (1:5000 dilution). -Actin migrated to 46 kDa. Ratios of the optical densities of IP3R-I, IP3R-II, and IP3R-III to the optical density of -actin were calculated.

Effects of CORT Pellet Implantation into Normal Rats on [³H]IP₃ Binding and on Immunolabeling of IP₃Rs. We determined B_max and K_D of [³H]IP₃ binding in cortex, hippocampus, and cerebellum after acute or chronic implantation of a CORT pellet into normal rats. Acute implantation of CORT (1 day) had no significant effects on B_max or K_D of [³H]IP₃ binding in any of the brain areas (data not shown). The results of B_max of [³H]IP₃ binding after 14 days of CORT pellet implantation are given in Table 2. It was observed that chronic implantation of CORT at both the 50- and the 100-mg dose significantly increased B_max of [³H]IP₃ binding in cortex, hippocampus, and cerebellum, and this effect was dose dependent, i.e., the magnitude of increase was greater with the 100-mg CORT pellet. Furthermore, CORT implantation, of either 50 or 100 mg, had no significant effects on K_D values in any of the brain areas studied (Table 2).

Effects of ADX and Simultaneous CORT Pellet Implantation into ADX Rats on IP₃ Levels. To examine whether the changes in IP₃ were responsible for the observed changes in [³H]IP₃ binding sites, we determined IP₃ levels in cortex, hippocampus, and cerebellum. One day after ADX there were no significant effects on the levels of IP₃ in cortex, hippocampus, or cerebellum. However, 14 days of ADX caused a substantial and significant increase in levels of IP₃ in cortex, hippocampus, and cerebellum (Table 3). Implantation of a 50- or 100-mg CORT pellet into ADX rats decreased the levels of IP₃ in all these brain areas. The 100-mg CORT pellet was able to completely prevent the ADX-induced increase in IP₃ levels in cortex, hippocampus, and cerebellum (Table 3).

Effects of CORT Pellet Implantation into Normal Rats on IP₃ Levels. IP₃ levels were measured in cortex, hippocampus, and cerebellum after acute and chronic implantation of a CORT pellet into normal rats. Acute (1-day) implantation of CORT had no significant effects on the levels of IP₃ in cortex, hippocampus, or cerebellum. When the levels of IP₃ were determined 14 days after CORT pellet implantation into normal rats, we observed that both the 50- and the 100-mg CORT pellet significantly decreased IP₃ levels in all the brain areas studied (Table 4). This effect was much more pronounced with the 100-mg dose than the 50-mg dose of CORT.

Effects of ADX and of CORT Pellet Implantation into ADX Rats and into Normal Rats on Levels of G_q/11  Proteins. Because G_q/11  protein has been shown to be coupled with PI-PLC (Strathmann and Simon, 1990), we determined whether ADX or CORT pellet implantation affects the levels of G_q/11  protein in rat brain. Acute or chronic ADX, or CORT pellet implantation into normal or ADX rats, did not cause any significant effects on the immunolabeling of G_q/11  protein in cortex or hippocampus (data not shown).

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

This investigation has led to the following observations: 1) bilateral ADX increased PI-PLC activity and, selectively, the mRNA and protein expression of only the PLC ₁ isozyme, and simultaneous CORT treatment of ADX rats prevented these changes; 2) CORT treatment of normal rats decreased PI-PLC activity and mRNA and protein levels of the PLC ₁ isozyme without any changes in the expression of PLC ₁ and ₁ isozymes; 3) ADX decreased the number of [³H]IP₃ binding sites, and CORT treatment of ADX rats reversed this decrease; 4) CORT pellet implantation increased [³H]IP₃ binding sites; 5) ADX or CORT treatment of ADX or normal rats had no significant effects on the immunolabeling of IP₃R isoforms; 6) ADX increased the levels of IP₃, whereas CORT treatment of ADX rats prevented this increase; 7) CORT treatment of normal rats decreased the levels of IP₃; and 8) ADX or CORT treatment had no significant effects on the expression of G_q/11  proteins. These results thus suggest that adrenal glucocorticoids affect various components of the PI signaling cascade in the central nervous system.

In a previous study, we demonstrated that chronic but not acute dexamethasone treatment of rats caused a significant increase in PI-PLC activity along with an increase in mRNA and protein levels of the PLC ₁ isozyme in cortex and hippocampus (Dwivedi and Pandey, 1999a). Interestingly, in the present study similar observations were noted in ADX rats, namely, ADX increased PI-PLC activity as well as mRNA and protein levels of the PLC ₁ isozyme in rat cortex and hippocampus. On the other hand, simultaneous CORT treatment of ADX rats dose dependently reversed these increases, and implantation of a CORT pellet into normal rats produced effects opposite to those elicited by ADX on PI-PLC activity and on mRNA and protein levels of the PLC ₁ isozyme. It thus appears that PI-PLC is under the regulation of endogenous glucocorticoids; however, why dexamethasone and CORT produce opposite changes is currently unclear. Some evidence suggests that dexamethasone penetrates poorly into the brain; however, uptake and retention of dexamethasone are very high in the pituitary (Birmingham et al., 1993). Recently, Meijer et al. (1998) reported that the multiple-drug-resistance gene's P-glycoprotein binds to dexamethasone and causes poor cell nuclear retention, and suggested that dexamethasone is a poor substitute for depleted endogenous glucocorticoids in brain. In view of these observations, it is possible that administration of dexamethasone might be inhibiting the release of CORT by a feedback mechanism; however, because dexamethasone penetrates poorly into the brain and does not substitute the endogenous glucocorticoids in brain, it may be producing effects similar to those of ADX.

Another important aspect is the selective action of CORT in regulating the expression of only the PLC ₁ isozyme. As is well known, PLC has been categorized into three major families: PLC , PLC , and PLC  (Cockcroft and Thomas, 1992). All PLC isozymes recognize PIP₂ as a substrate and carry out Ca²⁺-dependent hydrolysis of inositol lipids; however, these isozymes are differentially regulated and expressed (Cockcroft and Thomas, 1992). PLC  is activated by receptors that activate the G_q family of G proteins (Taylor et al., 1991), whereas PLC  is regulated by receptor and nonreceptor tyrosine kinases (Rhee and Bae, 1997). Little is known about the regulation of PLC . Our observation of altered expression of the PLC ₁ isozyme, without any changes in PLC ₁ or ₁, suggests that glucocorticoid action selectively affects the PI signal transduction system, in which PLC  is the principal mediator of PIP₂ hydrolysis. Furthermore, it appears that the decrease in PI-PLC activity could be due to a decrease in the expression of the PLC ₁ isozyme. This is supported by the notion that CORT treatment causes a generalized decrease in both membrane and cytosol PI-PLC activity, which suggests that this reduction in activity may be due to reduced synthesis of the PLC ₁ isozyme. In this context it is important to mention that the expression of G_q  protein remains unaltered, which supports the concept of selective action of glucocorticoids in brain.

IP₃ is one of the important second messengers produced from the hydrolysis of PIP₂ by the enzyme PI-PLC. The IP₃ signal is physiologically effective only on IP₃Rs, which transduce this intermediate signal to a Ca²⁺ signal, by mobilizing Ca²⁺ from intracellular stores, which then participate in various physiological functions (Berridge et al., 1998). To examine whether the decreases in activity and expression of the PLC ₁ isozyme had any further effects downstream, at the level of IP₃, we measured IP₃ levels, [³H]IP₃ binding to IP₃Rs, and the expression of individual IP₃R isoforms. It was observed that the number of [³H]IP₃ binding sites in cortex, hippocampus, and cerebellum was decreased by ADX, and that CORT treatment of ADX rats reversed these changes. Furthermore, CORT treatment of normal rats produced changes opposite to those elicited by ADX. Because IP₃ binds to all the IP₃R isoforms and because specificity and binding affinity of IP₃ do not differ in different classes of IP₃Rs (Yamada et al., 1994), we further examined whether the decrease in number of IP₃ binding sites was due to altered expression of IP₃R isoform(s). Three distinct types of the IP₃R family have been molecularly cloned: IP₃R-I, IP₃R-II, and IP₃R-III (Furuchi et al., 1994). IP₃R-I is the most characterized isoform (Ferris and Snyder, 1992; Mikoshiba, 1993) and is the predominant type in rodent brain. Our immunolabeling studies suggest that CORT treatment of normal or ADX rats has no significant effects on the protein expression of any of the IP₃R isoforms, which suggests that the decrease in [³H]IP₃ binding to IP₃R is not due to altered expression of IP₃R(s). However, it was observed that CORT treatment of normal rats decreased IP₃ levels, whereas ADX caused the opposite effect. Thus, it appears that the decrease in IP₃ levels by CORT could be the consequence of decreased PI-PLC activity, and that the number of binding sites for IP₃Rs could have been increased as a compensatory event in response to the decreased IP₃ levels. Another possibility could be that glucocorticoids may modify the activity of protein kinases, which may in turn cause altered phosphorylation of IP₃Rs, thereby alter the binding characteristics of IP₃Rs. In fact, it has been demonstrated that the binding characteristics of IP₃Rs are altered after phosphorylation mediated by protein kinase A (Snyder and Supattapone, 1989).

The mechanism(s) by which glucocorticoids cause changes in the expression of PLC ₁ and further downstream, at the level of IP₃, appears to be complex because both genomic and nongenomic actions of glucocorticoids have been proposed (Moore and Evans, 1999). Because we did not find significant changes in the expression of PLC ₁ after acute CORT treatment, whereas chronic treatment of CORT decreased its expression, it indicates that the action of CORT on PI-PLC is a delayed event and this effect may be indirect. There is a possibility that subsequent changes in the signaling cascade or in the brain, which could be genomic in nature, may be required to cause the alterations in the expression of PLC ₁. In this regard, it has been demonstrated that chronic administration of dexamethasone decreases noradrenaline-stimulated PI metabolism in rat brain (Takahashi et al., 1996). Furthermore, in the brain of aged rats, where glucocorticoids have been demonstrated to participate in aging (Landfield and Eldridge, 1991; Stein-Behrens and Sapolsky, 1992), it has been shown that ₁-adrenergic- and muscarinic receptor-stimulated PI hydrolysis is decreased (for review, see Bothmer and Jolles, 1994). Recently, Nicolle et al. (1999) have shown that glutamate receptor-mediated PI turnover is decreased in the brain of aged rats along with a decrease in the expression of PLC ₁. In addition, the expression of 5HT_2A and 5HT_2C receptors has been shown to be increased in rat brain after chronic glucocorticoid administration (Chaouloff, 1995). From these studies it appears that glucocorticoids cause an increase in the expression of receptors and a decrease in the receptor-mediated PI turnover. In the present study, we did not examine receptor-mediated IP₃ formation; however, in accordance with the previous studies, it appears that the decrease in IP₃ levels could be a consequence of decreased PI turnover in response to the decreased activity of PI-PLC and decreased expression of the PLC ₁ isozyme, which may have been desensitized in response to sustained stimulation of receptors and receptor-mediated PI hydrolysis. Further studies are needed to clarify these issues.

The functional implications of altered PI-PLC and IP₃ by glucocorticoids remain to be elucidated; however, as mentioned above, abnormal HPA function has been reported in affective disorders (Nelson and Davis, 1997). Furthermore, we have previously reported that the number of [³H]IP₃ binding sites is increased in platelets of depressed patients (Dwivedi et al., 1998), and that the expression of the PLC ₁ isozyme and PI-PLC activity are decreased in the prefrontal cortex of suicide subjects (Pandey et al., 1999). It is possible that these changes in depressed and/or suicide subjects could be related to abnormal HPA function. However, to fully understand the implications of altered PI-PLC and IP₃ in human mood and behavior and to elucidate the inter-relationship of altered HPA function with PI-PLC and IP₃, further clinical investigations need to be conducted.