Interaction between glucocorticoids and epidermal growth factor in vitro in the growth of palatal mesenchymal cells from the human embryo

Previous studies of ours have shown that palatal mesenchymal cells from the human embryo (HEPM cells) are responsive both to the glucocorticoid dexamethasone (DEX) and epidermal growth factor (EGF) through mechanisms associated with cytoplasmic and cell surface receptors, respectively. HEPM cell growth was inhibited by DEX and was stimulated by EGF. In the present study, the interactions between DEX and EGF were investigated. DEX (10(-6) M) enhanced EGF-stimulated HEPM cell growth as assessed by an increase in cell number and ornithine decarboxylase activity under serum-free cell culture conditions. DEX also enhanced the specific binding of 125I-EGF to these cells, which was reflected in an increase in both the number and the affinity of EGF receptors. EGF (1 ng/ml), on the other hand, decreased the number of sites per cell which specifically bind 3H-DEX. EGF completely prevented the inhibition by DEX of HEPM cell growth. These results indicated that DEX and EGF interact with each other in the process(es) regulating HEPM cell growth. This interaction may be partially influenced by direct modulation of existing receptors for DEX and EGF present in the cells.     

Down-Regulation of c-neu Receptors by Nerve Growth Factor in PC12 Cells

Abstract: A small number of p185^{c- neu} receptors have been found on PC12 cells. These receptors show some basal phosphorylation in quiescent cells. When the cells are treated with nerve growth factor (NGF) for a short time, some increase in phosphorylation is seen, mainly on serine and threonine residues, and this is accompanied by a slight shift in the apparent molecular weight. Epidermal growth factor (EGF) also increases the phosphorylation of p185^{c- neu} , in this case on tyrosine residues. Neither heregulin-β1 nor gp30 stimulates the tyrosine phosphorylation of p185^{c- neu} , and neither has a proliferative effect on the cells. Treatment of the cells with NGF for 5 days produces a 70–80% reduction in the number of p185^{c- neu} receptors. This down-regulation does not occur when PC12nnr5 cells, which lack the high-affinity NGF receptor, p140 ^trk , are treated with NGF.The level of p185^{c- neu} mRNA is not altered by NGF treatment, suggesting that the down-regulation is due to either a translational or a posttranslational alteration.     

Epidermal Growth Factor Promotes Uncoupling from Adenylyl Cyclase of the Rat D2S Receptor Expressed in GH4C1 Cells

Abstract: In anterior pituitary cells or when transfected into host cell lines, the D₂ dopamine receptor inhibits adenylyl cyclase and activates potassium channels. The GH-3 pituitary tumor cell line, which lacks functional D₂ receptors, responds to epidermal growth factor (EGF) by expressing a D₂ receptor that, paradoxically, couples to potassium channel activation but poorly inhibits adenylyl cyclase; this was correlated with a pronounced increase in α subunit of the G protein G₁₃. In this study we have investigated the effects of EGF on the transduction mechanisms of D₂ receptors in GH4C1 cells transfected and permanently overexpressing the rat short D₂ receptor. Activation of D₂ receptors in these cells resulted in both inhibition of adenylyl cyclase and opening of potassium channels and inhibition of prolactin release by both cyclic AMP-dependent and independent mechanisms. Exposure of the transfected GH4C1 cells to EGF caused a dramatic decrease in the coupling efficiency of the D₂ receptor to inhibit cyclic AMP-dependent responses, leaving its activity toward potassium channels unchanged. The EGF treatment led to the concomitant increase in the membrane content of G₁₃ protein. These results suggest that the transmembrane signaling specificity of G protein-coupled receptors can be modulated by the relative amounts of different G proteins at the cell membrane.     

Influence of Growth Hormone and Thyroxine On Cell Kinetics In the Proximal Tibial Growth Plate of Snell Dwarf Mice

Abstract. In Snell dwarf mice, the influence of short-term treatment with human growth hormone (hGH) or thyroxine on the proliferative and sulphation activity of the proximal tibial growth plate was studied. By autoradiographic methods, the [³H]methylthymidine incorporation after a single injection was measured, after 2 hr incorporation time. the labelling index was calculated and the number of labelled mitoses was counted. In addition, the distribution of the labelled nuclei over the proliferating and degenerating zones was determined by continuous labelling for 25 and 73 hr.
In untreated dwarf mice after [³H]-methylthymidine administration, the number of labelled nuclei in the growth plate is low. Labelling occurs, as expected, mainly in the cells of the proliferative zones. the number of labelled nuclei in control dwarf mice was similar after 25 and 73 hr continuous labelling. This suggests that many cells are in a resting G_o or prolonged G₁ phase. Both hGH and T₄ treatment induce a significant increase of the number of labelled nuclei per growth plate and of the number of mitoses. Since hormonal treatment induces a small number of mitoses after 2 hr incorporation of the label, the minimal G₂ phase of the cell cycle is less than 2 hr. In addition, treatment with hGH and T₄ stimulates chondrocytes in the zone of proliferative and hypertrophic cells to actively incorporate [³⁵S]-sulphate.     

Transferrin Receptor on Chick Fibroblast Cell Surface and the Binding Affinity in Relevance to the Growth Promoting Activity of Transferrin

We examined the transferrin (Tf) receptor of chick skin fibroblasts using chick ¹²⁵I-Tf. When the cells were incubated with ¹²⁵I-Tf on ice, most of the cell-associated ¹²⁵I-Tf was found on the cell surface; on the other hand, a large part of it was located inside the cells when incubated at 37°C. By equilibrium binding assay, the number of Tf receptors per cell was determined as 6.7 × 10³. Dissociation constant was estimated to be 2.6 × 10⁻⁸ M.
The binding of ¹²⁵I-Tf was competitively inhibited by the addition of chick unlabeled Tf. Weaker inhibition was observed when bovine Tf was used as a competitor. Horse Tf had no effect on the binding of chick Tf. This agrees well qualitatively with chick cell growth-promoting activites of these Tfs.
Removal of Fe from Tf affected the affinity for its receptors. About 5- to 10-fold higher concentrations of chick apo–Tf was needed to achieve the same degree of inhibition of ¹²⁵I-Tf binding as that made by chick Fe-Tf.     

Induction of a Nerve Growth Factor-Sensitive Kinase that Phosphorylates the DNA-Binding Domain of the Orphan Nuclear Receptor NGFI-B

Abstract: Nerve growth factor (NGF) induces the synthesis and the phosphorylation of the orphan nuclear receptor NGFI-B in PC12 cells. Previous work has shown that phosphorylation, by protein kinase A, of a specific serine in the DNA-binding domain inhibits its binding to the NGFI-B response element. Also, cytoplasmic extracts from PC12 cells phosphorylate this serine, and phosphorylation is greater in extracts from cells treated with NGF. The present work describes the induction, identification, and partial purification of a kinase (termed NGFI-B kinase I) from PC12 cell extracts that catalyzes this phosphorylation. Phosphorylation of the DNA-binding domain with this purified preparation inhibits its binding to the NGFI-B response element. The kinase is rapidly activated by treatment of the cells with NGF, and the activation lasts for at least several hours. It also is activated by fibroblast growth factor and epidermal growth factor (EGF), but the activation by EGF is quite transient. The kinase requires Mg²⁺ but will use Mn²⁺. The molecular mass of the kinase is 95–100 kDa, and it is different from protein kinase A, Fos kinase, or pp90 ^rsk . Comparison with a partially purified preparation of cyclic AMP response element-binding protein kinase, however, indicates that the two are either very similar or identical.     

In Human Fetal Astrocytes Exposure to Interleukin-1β Stimulates Acquisition of the GD3+ Phenotype and Inhibits Cell Division

Abstract: In human astrocyte cultures established from second-trimester fetal brain tissue, ∼5–10% of total astrocyte population in unstimulated cultures were GD3⁺/glial fibrillary acidic protein (GFAP)⁺. The GD3⁺ cells were always GFAP⁺ and grew as flat, highly spread cells but changed to process-bearing cells after interleukin-1β (IL-1β) stimulation. It is interesting that IL-1β, a known mitogen for rat astrocytes, suppressed human fetal astrocyte proliferation as determined by [³H]thymidine incorporation, bromodeoxyuridine (BrdU) labeling, and cell counting. The GD3⁺ population, however, consistently increased in absolute number after IL-1β stimulation, in a dose- and time-dependent manner. The IL-1β-mediated increase in number of GD3⁺ astrocytes was independent of initial cell density or serum concentration. By flow cytometry, IL-1β enhanced both the mean fluorescence intensity and the percentage of GD3⁺ cells. To investigate whether the increase in GD3⁺ astrocyte cell number was due to proliferation of preexisting GD3⁺ astrocytes or due to conversion of GD3⁻ to GD3⁺ cells, we performed BrdU/GD3 double immunocytochemistry. BrdU/GD3 double-positive cells were extremely rare in both control and IL-1β-stimulated cultures. Moreover, an increase in number of GD3⁺ astrocytes was still observed in control and IL-1β-stimulated cultures where GD3⁺ cells had been initially eliminated by cell sorting. These results indicate that GD3⁺ astrocytes in human fetal culture may represent a postmitotic, differentiated, distinct phenotype.     

Effect of protein and steroidal osteotropic agents on differentiation and epidermal growth factor-mediated growth of the CFK1 osseous cell line.

The effects of factors known to influence bone metabolism were examined using the osseous cell line CFK1. Parathyroid hormone (PTH) and dexamethasone (DEX) appeared to enhance the formation of cell foci of CFK1 cells in culture whereas retinoic acid (RA) caused a marked alteration in individual cell morphology. Bone morphogenetic protein (BMP-2) and PTH increased alkaline phosphatase activity, however, this index of differentiation was suppressed by epidermal growth factor (EGF), DEX, and RA. BMP-2 and EGF each stimulated DNA synthesis in a dose-dependent manner and enhanced cell numbers, but, no synergistic response of EGF and BMP-2 was observed. PTH and DEX failed to significantly alter cell number or EGF-stimulated DNA synthesis or cell proliferation. Although RA treatment of CFK1 cells resulted in a reduction in cell number compared to control, pretreatment with RA enhanced EGF-stimulated DNA synthesis and proliferative effects. At least part of this effect was by increasing the EGF receptor binding capacity of the cells. Furthermore, using cell cycle analysis, addition of EGF stimulated the progression of RA-treated cells into the DNA synthesis (S) phase with a reduced lag time. EGF and BMP-2, therefore, appear to exert a role in the expansion dynamics of the CFK1 population although BMP-2 may also enhance differentiation. PTH and DEX may act primarily to modulate the differentiated function of the CFK1 cells. RA inhibited cell proliferation and may mediate differentiation towards a less established cell population with upregulation of EGF receptors. The CFK1 cell model may, therefore, provide insight into microenvironmental control of growth and differentiation of precursor osseous cells.     

Growth Kinetics, Cell Shape, and the Cytoskeleton of Primary Astrocyte Cultures

Abstract: We examined correlations among growth kinetics, cell shape, and cytoskeletal protein content in rat astrocytes grown in primary culture. Cell suspensions from brains of newborn rats were seeded at densities from 0.2 to 3 × 10⁵/cm². At initial densities above 1 × 10⁵ the population increased to reach confluency by 10–12 days, after which cell number remained stable for many weeks. At low initial densities, 0.2–0.4 × 10⁵/cm², cells did not increase in number. Final density increased with increasing plating densities. High-density cells had small perikarya and several long cytoplasmic processes; low-density cells appeared flat and polygonal. All cultures were almost entirely astrocytic, as judged by immunofluorescent staining with antiserum against glial fibrillary acidic protein (GFAP). Cytoskeletal proteins were analyzed by gel electrophoresis after extraction from cells with nonionic detergent. Relative amounts of the proteins differed, in that low-density cells contained large amounts of cytoskeletal actin relative to the intermediate filament (IF) proteins vimentin and GFAP, whereas high-density cells contained relatively less actin and more IF proteins. Such differences in cytoskeletal proteins between the high- and low-density cultures were mirrored in the relative rates of synthesis of the cytoskeletal proteins. In the low-density cells amino acid incorporation into cytoskeletal-associated actin was more active than that into the IFs, whereas in the high-density cells higher rates of IF protein synthesis were observed.     

Effects of a Peptide Analogue of the Amphiphilic Domain of the Common Neurotrophin Receptor on Nerve Growth Factor-Mediated Motility of Human Neuroblastoma Cells

Abstract: Exposure of human neuroblastoma cells (IMR-32) to a peptide mimic of the cytoplasmic amphiphilic domain of the common neurotrophin receptor (p75^NTR 367–379) resulted in enhanced nerve growth factor (NGF)-mediated inhibition of cell invasion in vitro. The peptide also enhanced NGF-mediated neurite extension and GAP-43 gene expression but had no effect on NGF-mediated cell survival. These latter functional effects mimicked influences on NGF-mediated neurite growth in other trkA-positive cells as reported previously. NGF-dependent trkA phosphorylation was significantly enhanced by the presence of the peptide, whereas high-affinity binding of ¹²⁵I-NGF, both NGF receptors mRNA and protein expression, and trkA dimer/monomer ratios were not influenced. The studies suggest that ligand-mediated trkA activation has differential effects on cell motility phenomena and that the amphiphilic domain of p75^NTR has a role in this differential signaling.     

Nerve Growth Factor, Epidermal Growth Factor, and Insulin Differentially Potentiate ATP-Induced [Ca2+]i Rise and Dopamine Secretion in PC12 Cells

Abstract: To study how growth factors affect stimulus-secretion coupling pathways, we examined the effects of nerve growth factor (NGF), epidermal growth factor (EGF), and insulin on ATP-induced [Ca²⁺]_i rise and dopamine secretion in PC12 cells. After a 4-day incubation of cells, all three factors increased ATP-induced dopamine secretion significantly. We then examined which step of ATP-induced secretion was affected by the growth factors. Cellular levels of dopamine-β-hydroxylase and catecholamines were increased by NGF treatment but were not affected by EGF or insulin. The ATP-induced [Ca²⁺]_i rise was also enhanced after growth factor treatment. The EC₅₀ of ATP for inducing [Ca²⁺]_i rise and dopamine secretion was increased by NGF treatment but not by treatment with EGF or insulin. Accordingly, the dependence on [Ca²⁺]_i of dopamine secretion was increased significantly only in NGF-treated cells. Our results suggest that for EGF- and insulin-treated PC12 cells, the increase in secretion is mainly due to increased potency of ATP in inducing [Ca²⁺]_i rise. NGF treatment not only increased the potency of ATP but also decreased the Ca²⁺ sensitivity of the secretory pathway, which as a result becomes more tightly regulated by changes in [Ca²⁺]_i.     

Human Y-79 Retinoblastoma Cells Exhibit Specific Corticotropin-Releasing Hormone Binding Sites

Abstract: In this study we have identified specific binding sites for corticotropin-releasing hormone (CRH) in human Y-79 retinoblastoma cell membranes by using ¹²⁵I-Tyrovine CRH (¹²⁵I-oCRH) as radioligand. Binding at 19°C was rapid with steady state being reached within 20 min, reversible and linear with membrane protein concentration. The ¹²⁵I-oCRH binding was enhanced by Mg²⁺ and inhibited by the GTP analogue guanosine 5'- O -(3'-thiotriphosphate). Y-79 cell membranes exhibited two populations of binding sites, a high-affinity site with an apparent dissociation constant ( K _D) of 1 n M and a low-affinity site with an apparent K _D of 500 n M . ¹²⁵I-oCRH binding was completely antagonized by human/rat CRH, [Met(O)²¹]oCRH, α-helical CRH_9–41, urotensin I, and sauvagine with a rank order of potency similar to that displayed by CRH receptors of other tissues. These data describe for the first time the presence of specific CRH-binding sites in retinal cells. The Y-79 cell line may therefore constitute a valuable model in which to study CRH action on retinal cells.     

Generation Time and Growth Rate of the Human Intestinal Parasite Blastocystis hominis

ABSTRACT. Blastocystis hominis , an anaerobic intestinal protozoan parasite of man, has a generation time (GT) in axenic culture of 8.5–19.4 h, depending on the strain tested. Average GT of the eight strains was 11.7 h. Zero growth time cell counts of 5.0 × 10⁵/ml to 2.0 × 10⁶/ml rose in 3–5 days to 1 × 10⁷ or 1 × 10⁸ cells/ml. The GT was determined for the 24-h period during which the most rapid growth occurred; about 2% of the B. hominis cells were in division during this time. Division under the culture conditions provided was by binary fission, the usual mode for B. hominis in vitro as well as in vivo. Division times were determined also by direct observation of individual dividing cells in slide cultures. These were usually ca. 40–60 min but sometimes as low as 20 min.     

Tumour Cell Recruitment of the Jb-1 and L 1210 Ascites Tumour Determined Directly By Double Labelling With [14C]- and [3H]-Thymidine

Abstract. Tumour cell recruitment of the JB-1 and L 1210 ascites tumour has been demonstrated directly by a double-labelling method with [¹⁴C]- and [³H]-thymidine (TdR). After [¹⁴C]-labelling of all proliferating tumour cells by multiple injections of [¹⁴C]TdR, recruitment of resting cells was stimulated by removal of the majority of tumour cells, i.e. by maximum aspiration of ascitic fluid. the number of recruited resting cells in the remaining tumour that re-enter the cell cycle after stimulation was demonstrated directly by a single injection of [³H]TdR given at different times after stimulation. the increase in the percentage of purely [³H]-labelled cells, i.e. recruited cells, with increasing time after stimulation, shows that recruitment is not a synchronous but a continuous process, the maximum of which occurs earlier in the case of the L 1210 than the JB-1 tumour. This suggests that there seems to be a relationship between the time required for maximum recruitment and the corresponding cell cycle parameters of the unperturbed tumour. There is a transitory increase of the growth fraction to about 100% and a considerable shortening of the cycle time at the maximum of recruitment.     

Growth Fraction of Myelocytes In Normal Human Granulopoiesis

Abstract. The labelling index (LI) of myelocytes (M) after flash labelling of normal human bone marrow cells with [³H]-thymidine ([³H]TdR) is always lower that the LI obtained for myeloblasts (MB) and for promyelocytes (PM). This fact can be interpreted in two ways: it may mean that the duration of the G₁ phase of the cell cycle is longer in M than in MB or PM, or it may mean that the proportion of cells in cycle, i.e., the growth fraction (GF), is lower in the M population than in MB or PM. the evolution of the LI and of the mean number of grains per cell was monitored in [³H]TdR-labelled normal bone marrow during in vitro incubation for 50 hr. the generation time, measured by the halving time of the mean number of grains per cell after flash labelling, was similar for M to that for MB and PM. During continuous labelling, the LI of MB and PM reached 1 and the LI value for M never rose to more than 50% of the values recorded for MB and PM after 30 hr. These findings give support to the second hypothesis, i.e., a lower GF in the M population. Good correlation was found between the LI of M and the proportion of mature polymorphonuclear cells in the bone marrow of normal subjects and of patients with chronic benign neutropenia or hyperleucocytosis. Variations in the M growth fraction could be a medium-term (2-3 days) regulatory factor in granulocyte production.     

The Effects of Insect Juvenile Hormone on the Growth of Some Protozoans in vitro. I. Crithidia sp.

SYNOPSIS. Experiments were designed to investigate the effects of insect juvenile hormone (JH) on the over-all growth and macromolecular synthesis of Crithidia sp. in vitro. Cells grown in the presence of 10⁻⁵M-10⁻³M JH showed a concentration-dependent inhibition of growth, which appeared to result from both a prolongation of generation time and a delay in the onset of logarithmic growth. Juvenile hormone (10⁻³M) inhibited the incorporation of [³H]thymidine, [³H]uridine and [³H] leucine into logarithmically growing cells by 50, 70 and 40% respectively. The incorporation of [³H]uridine into acid insoluble material could be stopped within 1 hr of application of the hormone (10⁻³M). The inhibitory effect was reversible in terms of cell numbers in subcultures of washed cells but an examination of the reversibility of RNA synthesis inhibition suggested that the resumption of RNA synthesis at an optimal level would require a lag period of at least 1–3 hr. It is suggested that JH may act by interfering with RNA synthesis either directly or indirectly by primarily acting at the level of the plasma membrane.     

Involvement of Na+,K+-ATPase in the Mitogenic Effect of Insulin-Like Growth Factor-I on Cultured Rat Astrocytes

Abstract: We have previously reported that insulin/insulin-like growth factor (IGF)-I induced the α1 isoform of Na⁺,K⁺-ATPase in cultured astrocytes. In this study the effects of insulin/IGF-I on Na⁺,K⁺-ATPase activity and cell proliferation were examined in astrocytes cultured under the various conditions, to test the possible involvement of the enzyme activity in the mitogenic action of IGF-I on astrocytes. Insulin increased Na⁺,K⁺-ATPase activity and stimulated cell proliferation in subconfluent astrocytes (cultured for 7–14 days in vitro). In contrast, these effects were not observed in confluent cells (cultured for 28 days). Furthermore, insulin stimulated neither the enzyme activity nor [³H]thymidine incorporation in astrocytes preincubated in fetal calf serum-free medium for 2 days (quiescent cells) and treated with dibutyryl cyclic AMP (differentiated cells). The increases in Na⁺,K⁺-ATPase activity and expression of the α1 mRNA preceded the mitogenic effect. ¹²⁵I-IGF-I binding experiment showed that all the cells used here had similar binding characteristics. The insulin-induced increase in enzyme activity was not affected by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), and it was observed even in Ca²⁺-free medium. The stimulation by IGF-I of [³H]thymidine incorporation was attenuated by ouabain and a low external K⁺ level. These findings suggest that stimulation of Na⁺,K⁺-ATPase activity is involved in the mitogenic action of IGF-I on cultured astrocytes.     

Rapid Communication: cis-Methyldioxolane Specifically Recognizes the m2 Muscarinic Receptor

Abstract: cis -Methyldioxolane (CD) is a muscarinic receptor agonist. [³H] CD has been used to label a subpopulation of muscarinic receptors described as exhibiting high agonist affinity. Pharmacological evidence suggests that the population of receptors labeled by [³H] CD consists of m2 and/or m4 subtypes; however, no studies have directly addressed the subtype selectivity of [³H] CD. The present study characterizes binding of this ligand to individual human receptor subtypes expressed in transfected Chinese hamster ovary cells. Results indicate that [³H] CD binds with high affinity only to Hm2 receptors but not to all Hm2 receptors. Twenty-eight percent of Hm2 receptors bound [³H] CD with a K _D of 3.5 ± 0.5 nM. Binding was eliminated in the presence of guanosine 5'- O -(3-thiotriphosphate), indicating that the Hm2 receptors labeled by [³H] CD are those that are associated with GDP-bound G protein. Binding of [³H] CD by only a subpopulation of Hm2 receptors is in agreement with data generated from studies of [³H] CD binding in mammalian brain. Because muscarinic receptors have been implicated to play a role in the pathogenesis of both Alzheimer's and Parkinson's disease, as well as the neurotoxicity of organophosphorus compounds, knowledge of the binding specificity of the muscarinic agonist [³H] CD should aid research in these areas.     

Dexamethasone does not interfere with hormone-sensitive PI hydrolysis

Serum, but not epidermal growth factor (EGF), stimulated the release of radiolabeled inositol phosphates from human embryo palate mesenchyme (HEPM) cells prelabeled with [3H]-myoinositol. Pretreatment of cells with 10(-6) M dexamethasone (DEX) for 48 h had no effect on the release of inositol phosphates in response to serum. Furthermore, although treatment of the glucocorticoid-sensitive A/J strain of mouse embryo palate mesenchyme (MEPM) cells with 10(-6) M DEX inhibited their proliferation by 40%, it had no effect on the activity of phospholipase(s) C. However, DEX did enhance the incorporation of [3H]-myoinositol into membrane lipids. We interpret these data to mean that 1) serum factors enhance metabolism of inositol lipids in HEPM cells, 2) DEX does not interfere with the primary events by which agonists utilize metabolism of inositol lipids as a mechanism for transmembrane signaling, and 3) DEX may affect synthesis of phosphoinositides, as reported by Grove et al. (Biochem. Biophys. Res. Commun. 110:200-207, 1983; J. Craniofac. Genet. Dev. Biol. Suppl. 2:285-292, 1986).     

Administration of Dexamethasone Up-Regulates Protein Kinase C Activity and the Expression of γ and ε Protein Kinase C Isozymes in the Rat Brain

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 B _max and K _D 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 B _max 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.