Abbreviations used : DEX, dexamethasone ; ECL, enhanced chemiluminescence ; 5-HT, 5-hydroxytryptamine (serotonin) ; HPA, hypothalamic-pituitary-adrenal ; PDBu, phorbol 12, 13-dibutyrate ; PI, phosphatidylinositol ; PKC, protein kinase C ; TBST, 10 mM Tris base, 0.15 M NaCl, and 0.05% Tween 20.
Address correspondence and reprint requests to Dr. G. N. Pandey at Department of Psychiatry, University of Illinois at Chicago, 1601 West Taylor Street, Chicago, IL 60612, U.S.A.
Abstract : Altered hypothalamic-pituitary-adrenal (HPA) function (increased plasma cortisol level) has been shown to be associated with mood and behavior. Protein kinase C (PKC), an important component of the phosphatidyl-inositol signal transduction system, plays a major role in mediating various physiological functions. The present study investigates the effects of acute (single) and repeated (10-day) administrations of 0.5 or 1.0 mg/kg doses of dexamethasone (DEX), a synthetic glucocorticoid, on Bmax and KD of [3H]phorbol 12,13-dibutyrate ([3H]PDBu) binding, PKC activity, and protein expression of PKC isozymes, α, β, γ, δ, and ε in the membrane and the cytosolic fractions of rat cortex and hippocampus. It was observed that repeated administration of 1.0 mg/kg DEX for 10 days caused a significant increase in Bmax of [3H]PDBu binding to PKC, in PKC activity, and in expressed protein levels of the γ and ε isozymes in both the cytosolic and the membrane fractions of the cortex and the hippocampus, whereas a lower dose of DEX (0.5 mg/kg for 10 days) caused these changes only in the hippocampus. On the other hand, a single administration of DEX (0.5 or 1.0 mg/kg) had no significant effect on PKC in the cortex or in the hippocampus. These results suggest that alterations in HPA function from repeated administration of glucocorticoids may modulate PKC-mediated functions.
Earlier studies suggest that adrenal steroids are involved in various functional aspects of the CNS and may play an important role in the regulation of mood, behavior, emotion, and learning (McEwen et al., 1986 ; McEwen, 1987). The role of glucocorticoids in abnormal behavior is supported by observations of abnormalities in the hypothalamic-pituitary-adrenal (HPA) axis in depressed patients, which are reflected by a higher plasma cortisol level, increased levels of adrenocorticotropic hormone and corticotropin-releasing hormone in CSF, and failure to suppress plasma cortisol levels after treatment with dexamethasone (DEX), a synthetic glucocorticoid (Halbreich et al., 1985 ; Van de Kar, 1989 ; Murphy, 1991 ; Holsber et al., 1995). Further evidence comes from the finding that, in humans, glucocorticoid treatment induces depression (Ling et al., 1981). Recently, Fernandes et al. (1997) reported that chronic administration of glucocorticoids causes depression-like behavior in rats, suggesting that these behavioral changes may be associated with altered HPA function, induced by glucocorticoid hormones. The reason for the association between behavioral changes and HPA function is not clear. The reported interactions between glucocorticoids and serotonin [5-hydroxytryptamine (5-HT)] receptor subtypes, such as 5-HT1A, 5-HT1B, 5-HT2A, 5-HT2C (reviewed by Chauloff, 1995), and 5-HT7 (Le Corre et al., 1997 ; Shimuzu et al., 1997), and β-adrenergic (Kuroda et al., 1993) and glucocorticoid (Spencer et al., 1990) receptors suggest that the behavioral changes associated with abnormalities of the HPA axis may be due to alterations in these neurotransmitter receptors and in their functional responsiveness. This is supported by in vivo and in vitro studies, which suggest that glucocorticoids alter receptor-mediated signal transduction systems (Johnson and Jaworski, 1983 ; Rodan and Rodan, 1986 ; Duman et al., 1989 ; Robinson and Kendall, 1990 ; Akompong et al., 1993 ; Muraoka et al., 1993 ; Mitchell and Bansal, 1997).
Phosphatidylinositol (PI) hydrolysis is an important signal transduction pathway that is used by many receptors (Abdel-Latif, 1986). Agonists binding to these receptors cause the hydrolysis of PI 4,5-bisphosphate by activation of the enzyme phospholipase C, which generates two second messengers : inositol 1,4,5-trisphosphate and diacylglycerol. Inositol 1,4,5-trisphosphate mobilizes Ca2+ from intracellular sources, whereas diacylglycerol activates protein kinase C (PKC) (Nishizuka, 1988 ; Berridge and Irvine, 1989). PKC in turn phosphorylates many important proteins, which leads to cellular responses such as neuronal development, neurotransmitter release, and gene expression (Nishizuka, 1988). PKC is thus an important component of the PI signaling system. Although the interaction of glucocorticoids with 5-HT2A receptors (Kuroda et al., 1993 ; Pandey et al., 1995 ; Fernandes et al., 1997) and PI metabolism (Muraoka et al., 1993 ; Takahashi et al., 1996) has been studied, the postreceptor sites and other components of the PI signaling system and, more specifically, their interactions with glucocorticoids are not known. Some evidence suggests that PI metabolism and PKC level may be abnormal in depression (Mikuni et al., 1991 ; Karege et al., 1996 ; Pandey et al., 1998) ; however, it is not clear if an abnormal HPA axis can also cause changes in PKC.
To examine if an abnormal HPA axis is associated with altered PKC, we studied the effect of single and repeated administration of DEX on the density and the affinity of [3H]phorbol 12,13-dibutyrate ([3H]PDBU) binding to PKC, on PKC activity, and on immunolabeling of various PKC isozymes in rat brain.
MATERIALS AND METHODS
DEX, sesame oil, phorbol 12-myristate 13-acetate, phosphatidylserine, IgG, and polyethylene glycol were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). [3H]PDBu was obtained from New England Nuclear (Boston, MA, U.S.A.). [γ-32P]ATP and the PKC activity kit were purchased from Amersham (Arlington Heights, IL, U.S.A.). Monoclonal antibodies for PKC isozymes α, β, and γ were purchased from Sikagaku America (St. Petersburg, FL, U.S.A.), and PKC δ and ε isozyme antibodies were obtained from GibcoBRL (Gaithersburg, MD, U.S.A.). All other chemicals were of analytical grade and were purchased from Sigma Chemical Co.
Animals and treatment
Male Sprague-Dawley rats weighing 200-250 g were housed two per cage under standard light-dark conditions at a constant temperature (25°C). DEX was suspended in sesame oil, and subcutaneous injections were given to rats at doses of 0.5 or 1.0 mg/kg, either as a single acute dose or daily for 10 days. Control rats received the same volume of sesame oil subcutaneously. DEX-treated animals were provided normal saline [0.9% (wt/vol) NaCl] instead of water. The rats were decapitated 24 h after the last injection. The brains were removed quickly, and cerebral cortices and hippocampi were dissected out and immediately stored at -80°C until analysis.
The dose selection for DEX was based on the doses used by other investigators and our earlier study (Dwivedi et al., 1996) in which we observed that 0.5 and 1.0 mg/kg doses of DEX caused a significant increase in number of 5-HT2A receptors in the rat cortex. Whereas Duman et al. (1989) had found a dose-dependent increase in isoproterenol-stimulated cyclic AMP formation in the rat brain after administration of various doses of DEX (0.25-4 mg/kg), Kuroda et al. (1993) and Takahashi et al. (1996) had reported a significant increase in number of 5-HT2A receptors and a reduction in norepinephrine-stimulated PI metabolism, respectively, after administration of 1.0 mg/kg DEX to rats. Recently, Colangelo et al. (1998) had observed that treatment with DEX (0.5 mg/kg) caused an increase in content of nerve growth factor mRNA in the rat cortex and hippocampus.
Our experimental protocols were approved by the Animal Care Committee of the University of Illinois at Chicago.
[3H]PDBu binding to membrane and cytosolic PKC in rat brain
[3H]PDBu binding to membrane and cytosolic PKC was determined by a radioligand binding technique derived from a combination of the methods of Sharkey et al. (1984) and Gleiter et al. (1988). Similar procedures have been used in our laboratory to determine Bmax and KD of [3H]PDBu binding to PKC in membrane and cytosolic fractions obtained from the cortex of rats (Pandey et al., 1993) and from postmortem human brain tissues (Pandey et al., 1997). The procedure is as follows :
Preparation of membrane and cytosolic fractions.
Cortices and hippocampi were homogenized using a Polytron at a setting of 8 for 15 s in 10 volumes of homogenizing buffer (50 mM Tris-HCl, 2 mM EGTA, 1.0 mM MnCl2, and 1.0 mM phenylmethylsulfonyl fluoride). The homogenate was centrifuged at 100,000 g for 60 min at 4°C to separate the membrane fraction (pellet) from the cytosolic fraction. The pellet and the supernatant fraction were used to measure membrane and cytosolic PKC activity, respectively.
[3H]PDBu binding to membrane PKC.
The pellet obtained from the above procedure was resuspended in the required amount of incubation buffer [50 mM Tris-HCl (pH 7.4), 1.0 mM CaCl2, 75 mM magnesium acetate, 0.1% bovine serum albumin, and 50 μg/ml phosphatidylserine]. The binding assay was carried out in duplicate tubes containing the incubation buffer, [3H]PDBu ranging in concentration from 0.8 to 30 nM (six different concentrations), and 150 μl of membrane suspension with or without 10 μM phorbol 12-myristate 13-acetate in a total volume of 500 μl. The tubes were incubated for 30 min at 37°C. Bound [3H]PDBu was separated from free [3H]PDBu by addition of 5.0 ml of washing buffer [50 mM Tris-HCl (pH 7.4) containing 0.1% bovine serum albumin] and rapid filtration through a Whatman GF/B filter. Air-dried filters were used for liquid scintillation counting.
[3H]PDBu binding to cytosolic PKC.
The binding assays for the cytosolic fractions of the cortex and the hippocampus were carried out in duplicate tubes containing incubation buffer [50 mM Tris-HCl (pH 7.4), 1.0 mM CaCl2, 75 mM magnesium acetate, and 0.1% bovine serum albumin], 150 μl of the cytosolic fraction, [3H]PDBu (0.8-30 nM, six different concentrations), bovine γ-globulin (100 μg/ml), and phosphatidylserine (50 μg/ml) in a total volume of 500 μl. The tubes were incubated for 30 min at 37°C. The tubes were then chilled, and proteins were precipitated by addition of 200 μl of chilled 12% (wt/vol) polyethylene glycol (in 50 mM Tris-HCl, pH 7.4). To allow complete precipitation, the samples were kept for 15 min at 4°C. Bound [3H]PDBu was separated from free [3H]PDBu according to the methods described above.
The specific binding in the membrane and the cytosolic fractions was defined as the difference between the binding observed in the presence or absence of 10 μM phorbol 12-myristate 13-acetate. Bmax and KD were calculated by Scatchard analysis using the EBDA program (McPherson, 1985), and protein content was determined by the method of Lowry et al. (1951) using bovine serum albumin as the standard.
Determination of PKC activity in membrane and cytosolic fractions of rat brain
PKC activity in subcellular tissue fractions was measured according to the following procedure. The Amersham enzyme assay system was used to determine PKC activity (Mann et al., 1995), and a PKC-specific target peptide and all the necessary cofactors were provided in the kit. The tissue was homogenized in homogenizing buffer (50 mM Tris-HCl, 2 mM EGTA, and 5 mM EDTA) containing 2 mM dithiothreitol, 1.5 μM pepstatin, 2 μM leupeptin, 0.5 mM phenylmethylsulfonyl fluoride, and 0.2 U/ml aprotinin. The homogenate was centrifuged at 100,000 g for 60 min at 4°C. The supernatant was saved (cytosolic fraction), and the pellet was homogenized in homogenizing buffer containing 0.2% (wt/vol) Triton X-100. The homogenate was kept at 4°C for 60 min with occasional stirring and then centrifuged at 100,000 g for 60 min at 4°C. The resulting supernatant was used as the membrane fraction. Assay tubes (with a final incubation volume of 75 μl) contained 25 μl of a component mixture [3 mM Ca(C2H3O2)2, 75 μg/ml l-α-phosphatidyl-l-serine, 6 μg/ml phorbol 12-myristate 13-acetate, 225 μM substrate peptide, and 7.5 mM dithiothreitol in 50 mM Tris-HCl containing 0.05% sodium azide, pH 7.5] and 25 μl of the membrane or the cytosolic fraction. The reaction was initiated by addition of 25 μl of Mg-ATP buffer (10 μCi/ml [γ-32P]ATP, 1.2 mM ATP, 72 mM MgCl2, and 30 mM HEPES, pH 7.4) to each tube. The tubes were incubated for 15 min at 37°C, and the reaction was terminated by addition of 100 μl of the “stop” reagent (300 mM orthophosphoric acid containing carmosive acid) to each tube. An aliquot of the solution from each tube (35 μl) was blotted onto individual peptide-binding papers. Papers were washed with 75 mM phosphoric acid twice for 5 min. Papers were dried, and the retained radioactivity was counted by a liquid scintillation counter. The result was expressed as nanomoles per minute per milligram of protein. Before starting our experiments, the specificity of the PKC assay in the membrane and the cytosolic fractions of the cortex and the hippocampus was determined using staurosporine (100 nM) as the PKC inhibitor. It was observed that in the presence of staurosporine, PKC activity was 0.06% compared with the control.
Quantification of PKC isozymes in membrane and cytosolic fractions of rat brain by immunolabeling
Equal volumes of protein sample and gel loading solution [50 mM Tris-HCl (pH 6.8), 4% β-mercaptoethanol, 1% sodium dodecyl sulfate, 40% glycerol, and 1% bromphenol blue] were mixed, and the samples were boiled for 3 min and then kept on ice for 10 min. The samples (30 μg of protein in each lane) were loaded onto 7.5% (wt/vol) acrylamide gel using the Mini Protein II gel apparatus (Bio-Rad, Hercules, CA, U.S.A.). The gels were electrophoresed using 25 mM Tris base, 192 mM glycine, and 0.1% (wt/vol) sodium dodecyl sulfate at 150 V. The proteins were subsequently transferred electrophoretically to an enhanced chemiluminescence (ECL) nitrocellulose membrane (Amersham) using the Mini Trans Blot transfer unit (Bio-Rad) at a constant current of 0.150 A. The membranes were washed with TBST [10 mM Tris base, 0.15 M NaCl, and 0.05% (vol/vol) Tween 20] for 10 min. The blots were blocked by incubating with 5% (wt/vol) powdered nonfat milk in TBST, 2 ml of Nonidet P-40, and 0.02% (wt/vol) sodium dodecyl sulfate (pH 8.0). Then the blots were incubated overnight at 4°C with the primary monoclonal antibody (anti-PKC α, β, γ, δ, or ε) at a dilution of 1 : 3,000-1 : 5,000 (depending on the antibody used). The membranes were washed with TBST and incubated with horseradish peroxidase-linked secondary antibody (anti-rabbit IgG ; 1 : 3,000) for 3-5 h at room temperature. The membranes were extensively washed with TBST and exposed to ECL film. β-Actin was assayed simultaneously using the β-actin monoclonal antibody as the primary antibody and anti-mouse IgG as the secondary antibody. Before starting the experiments, the procedure used to determine the levels of PKC isozymes and β-actin was standardized using 10-100 μg of protein. We found that the bands were linear up to a concentration of 70 μg of protein. In addition, the dilution of antibodies and the duration of the exposure of nitrocellulose membranes on autoradiographic film were standardized. The bands on the autoradiogram were quantified using the Loats Image Analysis System, and the optical density of each sample was corrected using the optical density of the corresponding β-actin band. The values are represented as a percentage of the control.
To analyze the data, ANOVA was used, followed by Dunnett's test for multiple comparisons with the control group (Dunnett, 1980). Treatment groups were considered to be significantly different for p < 0.05. Data are mean ± SD values.
Effects of single and repeated administration of DEX on [3H]PDBu binding to membrane and cytosolic PKC in rat brain
It was observed that a single injection of DEX at either a 0.5 or a 1.0 mg/kg dose had no significant effect on Bmax or KD of [3H]PDBu binding to membrane or cytosolic PKC in either the cortex or the hippocampus (data not shown).
Data presented in Table 1 show that treatment with DEX at 1.0 mg/kg for 10 days caused a significant increase in Bmax of [3H]PDBu binding to membrane and cytosolic PKC in the cortex and the hippocampus, and the extent of this increase was significantly higher in the hippocampus than in the cortex. On the other hand, repeated administration of DEX at a lower dose (0.5 mg/kg) had no significant effect on Bmax of [3H]PDBu binding to membrane or cytosolic PKC in the cortex. In contrast, at this dose level, Bmax of [3H]PDBu binding was significantly increased in both the membrane and the cytosolic fractions of the hippocampus.
Table 1. Effects of repeated administration of DEX (0.5 or 1.0 mg/kg for 10 days, 24 h after the last injection) on [3H]PDBu binding to PKC in rat brainData are mean ± SD values. Bmax is given as pmol/mg of protein ; KD is given as nM.DEX-treated groups were compared with the control group :
There were no significant differences in KD of [3H]-PDBu binding to membrane or cytosolic PKC in the cortex or the hippocampus between the controls and the DEX-treated groups.
Effects of single and repeated administration of DEX on PKC activity in rat brain
PKC activity was determined in the membrane and the cytosolic fractions obtained from cortical and hippocampal brain regions. As we observed in the case of [3H]-PDBu binding to PKC, a single administration of a 0.5 or a 1.0 mg/kg doses of DEX to rats had no significant effect on PKC activity in either the membrane or the cytosolic fractions obtained from the cortex or the hippocampus (data not shown). In contrast, repeated administration of DEX at 1.0 mg/kg significantly increased PKC activity in both the membrane and the cytosolic fractions of the cortex (Fig. 1A) and the hippocampus (Fig. 1B). The extent of increase in PKC activity was higher in the hippocampus than in the cortex. We did not observe any significant effect of DEX at the lower dose (0.5 mg/kg) on PKC activity in the cortex (Fig. 1A) ; however, PKC activity was increased in both the membrane and the cytosolic fractions of the hippocampus at this dose level (Fig. 1B).
Effects of single and repeated administration of DEX on expressed levels of PKC isozymes in rat brain
The steady-state concentrations of the levels of protein for the α, β, γ, δ, and ε isozymes of PKC in both the membrane and the cytosolic fractions of rat cortex and hippocampus were determined after single and repeated administrations of DEX (0.5 or 1.0 mg/kg). Representative autoradiograms of PKC α, β, γ, δ, and ε proteins are shown in Fig. 2. The apparent molecular masses for PKC α, β, and δ isozymes were 80 kDa, whereas PKC γ and ε migrated to 77.6 and 90 kDa, respectively. These molecular masses are consistent with those reported in the literature (Olivier and Parker, 1991 ; Koide et al., 1992 ; Hug and Sarre, 1993). To normalize our data, we simultaneously measured levels of β-actin. The apparent molecular mass of the β-actin protein was 46 kDa. The optical density of each band was corrected by the optical density of the corresponding β-actin band on the same immunoblot. This procedure has been used earlier in our laboratory (Dwivedi and Pandey, 1997).
We did not observe any significant effects after a single administration of DEX (0.5 or 1.0 mg/kg) on steady-state levels of protein for PKC α, β, γ, δ, or ε isozymes in either the membrane or the cytosolic fractions obtained from cortical or hippocampal brain regions (data not shown). Representative autoradiograms of the levels of protein for PKC isozymes after repeated administration of DEX are provided in Fig. 2A (cortex) and B (hippocampus). When the immunologically detectable levels of protein for PKC isozymes (α, β, γ, δ, and ε) were determined after repeated administration of a 1.0 mg/kg dose of DEX, a significant increase in steady-state levels of protein for PKC γ and ε isozymes was observed in both the membrane and the cytosolic fractions of the cortex (Fig. 3) and the hippocampus (Fig. 4). Repeated administration of the 0.5 mg/kg dose of DEX significantly increased levels of PKC ε and γ isozymes in both the membrane and the cytosolic fractions of the hippocampus (Fig. 4B) but had no significant effect on the levels of protein for PKC isozymes in either the membrane (Fig. 3A) or the cytosolic (Fig. 3B) fraction of the cortex. Repeated administration of 0.5 or 1.0 mg/kg doses of DEX did not cause any significant changes in content of PKC α, β, or γ isozymes in either brain region. To validate the data, we initially determined the immunolabeling of each PKC isozyme using five different concentrations of protein from control and DEX-treated rat brain. It was observed that the optical density was linear with the increased concentration of protein and that the curve was shifted toward the left in samples in which the immunolabeling of PKC γ and ε was increased.
The important findings of the present study are that repeated administration of DEX at 1.0 mg/kg caused significant increases in Bmax of [3H]PDBu binding to PKC, PKC activity, and level of protein expression of PKC γ and ε isozymes in both the membrane and the cytosolic fractions of rat cortex and hippocampus. On the other hand, a lower dose (0.5 mg/kg) of DEX significantly increased Bmax of [3H]PDBu binding to PKC, PKC activity, and immunolabeling of PKC γ and ε isozymes in both the membrane and the cytosolic fractions of rat hippocampus but not of cortex. A single administration of DEX (0.5 or 1.0 mg/kg) failed to change [3H]PDBu binding to PKC, PKC activity, or protein expression of PKC α, β, γ, δ, and ε in either brain region.
The isoforms of PKC constitute a family of Ca2+- or phospholipid-dependent serine/threonine kinases. Structurally, PKC consists of a regulatory domain (containing a pseudosubstrate sequence, cysteine-rich regions, and an NH2 terminus) and a catalytic domain (containing the carboxy terminus and binding sites for ATP and substrate proteins) (Nishizuka et al., 1991). [3H]PDBu binds to the regulatory domain of PKC (Parker et al., 1986), whereas the catalytic activity of PKC can be determined by measuring the transfer of the γ-phosphate group of ATP to the peptide specific for PKC. Our results demonstrate that PKC activity and Bmax of [3H]PDBu binding are increased in the membrane and the cytosolic fractions of rat cortex and hippocampus after repeated administration of DEX. This suggests that repeated administration of DEX modulates the regulatory as well as the catalytic domains of PKC.
Because [3H]PDBu binding to PKC is unable to discriminate among the various isozymes of PKC (Dimitrijevic et al., 1995), it is important to examine if this increase in Bmax of [3H]PDBu binding and the increase in PKC activity in the membrane and the cytosolic fractions of rat cortex and hippocampus are associated with any changes in expression of specific PKC isozymes. At least 12 PKC isozymes are known. They are classified as conventional (α, βI, βII, and γ), novel (δ, ε, ηθ, and μ), or atypical (ξ, λ, and ι). Novel PKCs differ from conventional PKCs because they do not require Ca2+ for activation, and they can be differentiated from atypical PKC isozymes, which are also Ca2+-independent but are insensitive to diacylglycerol and to phorbol ester activation. Each PKC isozyme differs in its distribution, biochemical characteristics, and substrate specificity. PKC isozymes α, γ, and ξ are present in nearly all tissues, whereas PKC ε, η, and θ are restricted to a few tissues, and PKC γ is present only in the brain and the spinal cord (Nishizuka, 1988 ; Nishizuka et al., 1991 ; Casabona, 1997). When we determined the expressed levels of PKC isozymes α, β, γ, δ, and ε, we observed that repeated but not single administration of DEX selectively increased the levels of only γ and ε isozymes, which suggests that the increase in [3H]PDBu binding and in PKC activity is associated with an increase in the expression of PKC γ and ε isozymes, specifically.
The molecular mechanisms of glucocorticoid action on PKC seem complex, as several neurotransmitters and neurotransmitter receptors, such as 5-HT1A, 5-HT1B, 5-HT2A, 5-HT2C, GABA, and glucocorticoid receptors, are affected by glucocorticoids. Thus, it is not clear whether the increase of PKC activity in rat brain after DEX administration depends primarily on glucocorticoid receptor activation. In this context, it is important to note that chronic administration of DEX decreases norepinephrine-stimulated PI hydrolysis in rat cortex (Takahashi et al., 1996), which suggests that, following longterm administration, the effect of DEX may be indirect, owing to changes in multiple components of the HPA axis caused by negative feedback inhibition. This is supported by the fact that a single administration of DEX had no significant effect on PKC in either the cortex or the hippocampus.
From our results, it appears that the cortex is relatively insensitive to the effect of DEX in comparison with the hippocampus. In the present study we found that in the cortex, repeated administration of DEX at the lower dose (0.5 mg/kg) had no significant effects on PKC but that the higher dose (1.0 mg/kg) not only increased [3H]-PDBu binding to PKC but also increased PKC activity and the protein expression of specific PKC isozymes, namely, γ and ε. In contrast, in the hippocampus, repeated administration of both the lower and the higher dose of DEX significantly increased these same parameters, but the extent of the increase with the 0.5 mg/kg dose was almost equal to that with the 1.0 mg/kg dose. Thus, in the hippocampus, where nearly equal effects were seen in response to doses of 0.5 or 1.0 mg/kg, the maximal effect may already have been obtained at the lower dose.
The reasons for the differential effects of DEX in the cortex and in the hippocampus are unclear ; however, this may indicate that the effect of DEX on PKC may be indirect, via the glucocorticoid receptors. Glucocorticoid receptor numbers are higher in the hippocampus than in other brain regions (Fuxe et al., 1985), and this may explain why lower doses of DEX suffice to produce a significant response in the hippocampus. It is also possible that the coupling between glucocorticoid receptors and the PI signaling system, which involves PKC, may be more efficient in the hippocampus.
The functional implications of the DEX-induced increase in PKC expression remain to be elucidated ; however, because hyperactive HPA function (increased cortisol level) has generally been reported in major depression (Halbreich et al., 1985 ; Maes et al., 1991, 1993) and because PKC has recently been shown to be abnormal in psychiatric illnesses such as major depression (Friedman et al., 1993) and suicide (Pandey et al., 1998), it is possible that changes in PKC activity may be caused by altered HPA function. This possibility is supported by our recent observation that [3H]PDBu binding to PKC is increased in platelets of depressed patients (Pandey et al., 1998). The abnormal PKC levels observed in depression, possibly caused by altered HPA function, may be associated with certain abnormal behavioral functions ; however, the interrelationship between the HPA and PKC in clinical situations needs to be investigated further so as to understand fully the implications of altered PKC in depression and suicide.
In summary, the present study demonstrates that repeated administration of DEX increases the number of the catalytic and the regulatory subunits of PKC, along with increasing the gene expression of PKC γ and ε isozymes. This suggests that the alteration of HPA function by long-term administration of glucocorticoids may result in modulation of PKC-mediated functions. The functional consequences of such an increase in PKC activity, i.e., the phosphorylation of specific PKC substrates, are under investigation.