Homologous Desensitization of the D1 Dopamine Receptor

Preincubation of D384 cells, derived from the human astrocytoma cell line G-CCM, with dopamine resulted in a time-dependent attenuation of cyclic AMP responsiveness to subsequent dopamine stimulation. This effect was agonist specific because the prostaglandin E1 (PGE1) stimulation of cyclic AMP of similarly treated cells remained unchanged. The attenuation by dopamine was concentration dependent with a maximum observed at 100 microM. A comparison of dopamine concentration-response curves of control and dopamine-preincubated cells revealed no change in the Ka apparent value, but a marked attenuation of the maximal response. Preincubation of cells with dopamine in the presence of D1 but not D2 selective antagonists partially prevented the observed attenuation. Attenuations in dopamine responsiveness were also obtained when D384 cells were preincubated with D1 but not D2 receptor agonists. The level of attenuation attained related to agonist efficiency in stimulating cyclic AMP: SKF38393 less than 3,4-dihydroxynomifensine less than fenoldopam less than 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene = dopamine. However, increasing the efficiency of 3,4-dihydroxynomifensine stimulation of cyclic AMP, using the synergistic effect of adding a low concentration of forskolin, produced no further change in the attenuation of the subsequent response to dopamine. Thus, the D1 dopamine receptors expressed by D384 cells undergo homologous desensitization. Uncoupling of the D1 dopamine receptor appears to be independent of cyclic AMP formation, analogous to a mechanism proposed for the beta-adrenergic receptor.     

D-1 Dopaminergic and β-Adrenergic Stimulation of Adenylate Cyclase in a Clone Derived from the Human Astrocytoma Cell Line G-CCM

Clones have been isolated from the human astrocytoma cell line G-CCM. Homogenates of clone D384 contain an adenylate cyclase that is stimulated by 3,4-dihydroxyphenylethylamine (dopamine), noradrenaline, and isoprenaline with Ka apparent values of 4, 56, and 2.7 microM, respectively. The Ka apparent value for dopamine was increased by the D-1 antagonist cis-flupenthixol, 25 and 100 nM, to 23 and 190 microM, respectively, but was unaffected by propranolol (1 microM). Noradrenaline stimulation of adenylate cyclase was only partially inhibited by either propranolol (10 microM) or cis-flupenthixol (1 microM). Propranolol (10 microM), but not cis-flupenthixol (1 microM), prevented stimulation by isoprenaline. The stimulation of adenylate cyclase by dopamine and noradrenaline remained unchanged in the presence of phentolamine (1 microM) and sulpiride (1 microM). These results suggest that clone D384 contains both D-1 dopaminergic and beta-adrenergic receptors coupled to adenylate cyclase. Dopamine stimulates D384 adenylate cyclase through D-1 receptors, isoprenaline via beta-receptors, and noradrenaline through both receptors.     

Characterization of Dopamine and β-Adrenergic Receptors Linked to Cyclic AMP Formation in Intact Cells of the Clone D384 Derived from a Human Astrocytoma

3,4-Dihydroxyphenylethylamine (dopamine) and beta-adrenergic receptor agonists and antagonists were assessed for their effects on cyclic AMP accumulation in human astrocytoma derived clone D384 cells. Dopamine, SKF 38393, and 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene increased cyclic AMP content with Ka values of 2.0, 0.2, and 1.6 microM. The D1-selective antagonists SCH 23390 (Ki, 1.2 nM) and SKF 83566 (Ki, 0.8 nM) were over 5,000-fold more potent than the D2-selective antagonist domperidone (Ki, 6.7 microM) at inhibiting dopamine stimulation of cyclic AMP formation. SCH 23388 (Ki, 560 nM; the S-enantiomer of SCH 23390) was 400-fold less potent than SCH 23390. Isoprenaline, adrenaline, salbutamol, and noradrenaline increased cyclic AMP content with Ka values of 0.13, 0.12, 0.22, and 7.60 microM. The beta 2-selective antagonist ICI 118,551 (Ki,0.8 nM) was almost 8,000-fold more potent than the beta 1-selective antagonist practolol (Ki, 5.9 microM) at inhibiting isoprenaline stimulated cyclic AMP accumulation. These results demonstrate that D384 cells express D1-dopamine and beta 2-adrenergic receptors linked to adenylate cyclase. Furthermore, the dopamine receptor expressed by D384 cells exhibits a pharmacological profile typical of a mammalian striatal D1-receptor and therefore the use of this clone represents another approach to studying central D1-receptors.     

D1 Dopamine Receptors of NS20Y Neuroblastoma Cells Are Functionally Similar to Rat Striatal D1 Receptors

Dopamine or agonists with D1 receptor potency stimulated cyclic AMP (cAMP) accumulation in whole cell preparations of NS20Y neuroblastoma cells. The accumulation of cAMP after D1 stimulation was rapid and linear for 3 min. Both dopamine and the novel D1 receptor agonist dihydrexidine stimulated cAMP accumulation two- to three-fold over baseline. The pseudo-Km for dopamine was approximately 2 microM, whereas for dihydrexidine it was approximately 30 nM. The effects of both drugs were blocked by either the D1-selective antagonist SCH23390 (Ki, 0.3 nM) or the nonselective antagonist (+)-butaclamol (Ki, 5 nM). Both (-)-butaclamol and the D2-selective antagonist (-)-sulpiride were ineffective (Ki greater than 3 microM). Forskolin (10 microM), prostaglandin E1 (1 microM), and adenosine (10 microM) also stimulated cAMP accumulation, but none were antagonized by SCH23390 (1 microM). Finally, muscarinic receptor stimulation (100 microM carbachol) inhibited both D1- and forskolin-stimulated increases in cAMP accumulation by 80%. The present results indicate that NS20Y neuroblastoma cells have D1 receptors that are coupled to adenylate cyclase, and that these receptors have a pharmacological profile similar to that of the D1 receptor(s) found in rat striatum.     

Glucocorticoids Enhance Histamine-Evoked Catecholamine Secretion from Bovine Chromaffin Cells

Abstract: The synthetic glucocorticoid dexamethasone enhanced histamine-evoked catecholamine secretion from cultured bovine chromaffin cells. Dexamethasone enhanced the effects of histamine on both adrenergic (epinephrine-rich) and noradrenergic (norepinephrine-rich) chromaffin cells but had a more dramatic effect on noradrenergic cells. Histamine-evoked secretion in noradrenergic cells appeared to become rapidly inactivated, whereas the rate of secretion in adrenergic cells was nearly constant for up to 2 h; dexamethasone treatment attenuated the inactivation seen in noradrenergic cells. The effect of dexamethasone appeared after a lag of several hours and was maximal by 24 h. The EC₅₀ for dexamethasone was ∼1 n M . The effect of dexamethasone was mimicked by the glucocorticoid agonist RU 28362 and was blocked by the antagonist RU 38486, indicating that the effects of these steroids were mediated by the glucocorticoid or type II corticosteroid receptor. Histamine-evoked catecholamine secretion in both dexamethasone-treated and untreated cells was blocked by the H₁ histamine receptor antagonist mepyramine but was not affected by the H₂ antagonist cimetidine; thus, dexamethasone appeared to enhance an H₁ receptor-mediated process. In the absence of glucocorticoids, H₁ receptor mRNA levels were higher in adrenergic than in noradrenergic cells. Dexamethasone increased H₁ receptor mRNA levels in both cell types. The increased expression of H₁ receptors presumably contributes to the enhancement of histamine-evoked catecholamine secretion by glucocorticoids. Glucocorticoids may play a physiological role in modulating the responsiveness of chromaffin cells to histamine and other stimuli.     

Glucocorticoids Potentiate the Prostaglandin E1-Mediated Cyclic AMP Formation by a Cultured Murine Neuroblastoma Clone

The effect of steroid hormones on the prostaglandin E1 (PGE1)-mediated cyclic AMP formation by murine neuroblastoma clone N1E-115 was studied. Dexamethasone at submicromolar concentrations and corticosterone at micromolar concentrations (steroids with glucocorticoid activity) were able to modify the PGE1-mediated response whereas testosterone, progesterone, and estradiol each at 10 microM had no effect. Glucocorticoids added to the culture medium of N1E-115 cells produced an increase in the maximal response to PGE1 only after long-term (greater than or equal to 4 h) incubation with the hormone. Inhibitors of protein and RNA synthesis blocked this effect of glucocorticoids. Basal activity of adenylate cyclase in treated cells was twofold higher than that in control cells, and this enzyme seemed to be the primary target for the hormone action, since the activity of 3':5'-cyclic AMP phosphodiesterase and the binding of [3H]PGE1 to its receptors were not altered by glucocorticoid treatment. Our results indicate that glucocorticoids modulate receptor-mediated responses in cells of neural origin through a mechanism that involves induction of protein synthesis.     

Hormonal alteration of methotrexate and folate polyglutamate formation in cultured hepatoma cells

Glutamylation of the antifolate methotrexate in H35 hepatoma cells was stimulated by physiologic concentrations of insulin and dexamethasone. At saturating concentrations of the hormone a 2.7-fold stimulation could be obtained with insulin (65 nM, 16-h exposure) and a 1.8-fold stimulation with dexamethasone (100 nM, 16-h exposure). The increases in glutamylation caused by the hormones were not additive, and both were inhibited by actinomycin D and cycloheximide. N6,O2'-dibutyryl cAMP and theophylline caused a modest reduction of glutamylation in control and dexamethasone-treated cultures, but repressed the stimulation caused by insulin by approximately one-third. Enhancement of synthesis by dexamethasone and insulin was associated with increases in the tri-, tetra-, and pentaglutamate derivatives of methotrexate, with little change in intracellular methotrexate and methotrexate diglutamate. When the conversion of folinic acid into the folylpolyglutamate pool was examined in folate-depleted H35 cells, insulin and dexamethasone had similar effects. The results suggest that these hormones play a role in the glutamylation of the folate coenzymes in a liver-derived transformed cell line in culture and that these effects are also reflected in the interaction of the cells with antifolates such as methotrexate.     

Dexamethasone stimulates arachidonic acid conversion to prostaglandin E2 in human amnion cells

The purpose of this investigation was to study the mechanism of stimulation of PGE2 output from human amnion epithelial cells by the synthetic glucocorticoid dexamethasone. Cells incubated in serum-free pseudo-amniotic fluid produced very low levels of PGE2, even when arachidonic acid (1 microM) was present. Pretreatment of cells with dexamethasone (50 nM) for 21 h increased the PGE2 output 6- to 7-fold in 2-h incubations only in the presence of arachidonic acid. The RNA synthesis inhibitor, actinomycin D (1 microgram/ml), and the protein synthesis inhibitor, cycloheximide (40 micrograms/ml), each blocked dexamethasone-stimulated arachidonic acid conversion to PGE2. The time course of these events suggests that dexamethasone first initiates RNA synthesis. Acetylsalicylic acid, a specific and irreversible blocker of prostaglandin endoperoxide H synthase (cyclooxygenase), was used to determine whether dexamethasone could stimulate new enzyme synthesis. Cells treated first with acetylsalicylic acid (30 min) then dexamethasone (22 h) produced as much PGE2 in response to 1 microM arachidonate as did cells exposed to dexamethasone only. Exposing cells to acetylsalicylic acid after dexamethasone completely eliminated PGE2 output. These data suggest that dexamethasone stimulates the synthesis of prostaglandin endoperoxide H synthase.     

Specific Inhibition of Dopamine D-1-Mediated Cyclic AMP Formation by Dopamine D-2, Muscarinic Cholinergic, and Opiate Receptor Stimulation in Rat Striatal Slices

The ability of different receptors to mediate inhibition of cyclic AMP accumulation due to a variety of agonists was examined in rat striatal slices. In the presence of 1 mM 3-isobutyl-1-methylxanthine, dopamine D-2, muscarinic cholinergic, and opiate receptor stimulation by RU 24926, carbachol, and morphine (all at 10(-8)-10(-5) M), respectively, inhibited the increase in cyclic AMP accumulation in slices of rat striatum due to dopamine D-1 receptor stimulation by 1 microM SKF 38393. In contrast, these inhibitory agents were unable to reduce the ability of a number of other agonists, including isoprenaline, prostaglandin E1, 2-chloroadenosine, vasoactive intestinal polypeptide, and cholera toxin, to increase cyclic AMP levels in striatal slices. These results suggest that in rat striatum either dopamine D-2, muscarinic cholinergic, and opiate receptors are only functionally linked to dopamine D-1 receptors or that the D-1 and D-2 receptors linked to adenylate cyclase lie on the cells, distinct from other receptors capable of elevating striatal cyclic AMP levels.     

Long-term treatment of swiss 3T3 fibroblasts with dexamethasone attenuates MAP kinase activation induced by insulin-like growth factor-I (IGF-I)

Bone formation is reduced in hyperglucocorticoid states, e.g. Cushing's syndrome or long-term treatment with synthetic glucocorticoids during rheumatic diseases. Possibly related to decreased sensitivity of the target to insulin-like growth factor-I (IGF-I). In this study, we have sought to identify postreceptor-mechanisms for glucocorticoid-induced resistance to insulin-like peptides in a model system. Treatment of Swiss 3T3 fibroblasts with 100 nM dexamethasone for 48 h reduced IGF-I-induced activation of mitogen-activated protein kinase (MAP kinase). The level of insulin receptor substrate-1 (IRS-1) was reduced in dexamethasone-treated cells, as measured by Western blot; however, the pattern of tyrosine-phosphorylated protein subsequent to stimulation with IGF-I (1 min) was not altered. No inhibitory effect of dexamethasone was observed on the level of phosphotyrosine in IRS-1 in extracts from IGF-I-treated cells. The amount of IGF-I-induced association of insulin receptor substrate-1 and phosphatidylinositol 3-kinase was increased in steroid treated cells. Addition of IGF-I increased the synthesis of lipid, glycogen and protein, and the reduction of a tetrazolium dye, MTS, in untreated cells. The response to IGF-I in terms of glycogen synthesis was blunted, whereas the effect of IGF-I was unaffected for the other three parameters in cells pretreated with dexamethasone. These findings indicate that the activation of MAP kinase may be dissociated from IGF-I-induced anabolic pathways and tyrosine phosphorylationof IRS-1. The results agree with the previously proposed role for the activation of MAP kinase in the regulation of glycogen synthesis. Furthermore, they suggest that dexamethasone-induced reduction of IRS-1 expression may be important for the impaired activation of MAP kinase by insulin-like peptides in steroid-treated cells.     

Role of Neostriatal Dopamine D1 and D2 Receptors in the Organization of Neuronal Activity in the Entopeduncular Nucleus

In chronic experiments on cats, we studied the effects of selective blockade of the dopamine D1 and D2 receptors in the nucleus caudatus (NC) against the background neuronal activity (BA) in the entopeduncular nucleus (ENT) and on the responses of these neurons evoked by stimulation of the subthalamic nucleus (STN). A blocker of D1 receptors, SCH 23390, and sulpiride, a blocker of D2 receptors, when injected into the NC head, led to changes in the BA frequency. The direction of these effects was different: blockade of the D2 receptors evoked a significant decrease in the BA frequency of ENT neurons, but when the D2 receptors were blocked, a trend toward an increase in this frequency was observed. Some of the ENT cells demonstrated similar changes in the temporal organization of their impulse activity with blockade of the D1 and D2 receptors. Namely, besides single action potentials (AP), high-frequency burst discharges began to be generated. Blockade of both D1 and D2 receptors prolonged the responses of ENT neurons to STN stimulation due to the appearance of similar AP bursts, while control animals demonstrated reactions consisting of solely single AP. The question on a dual (synergic and antagonistic) involvement of the neostriatal D1 and D2 receptors in the dopaminergic regulation of the basal ganglia is under discussion.     

Dexamethasone induces irreversible G1 arrest and death of a human lymphoid cell line.

Growth of a human leukemic T-cell line (CEM C7) in 10(-6) M dexamethasone results in inhibition of growth and rapid loss of cell viability after a delay of approximately 18 to 24 hours. Analysis of dexamethasone-treated cells by flow-microfluorometry showed that they were arrested in the G1 phase of the cell cycle. Loss of cell viability began at the same time as G1 accumulation was first detectable, and 20% of all cells were found to be blocked in G1 at this time suggesting that loss of viability and G1 arrest were coincident events. Half-maximal and maximal effects on both viability and G1 arrest after 48 hours in steroid were nearly identical with respect to steroid concentration and corresponded to half-maximal and full occupancy of glucocorticoid specific receptor by hormone, consistent with a glucocorticoid receptor mediated mechanism for both phenomena. Most non-viable cells were arrested in G1, and accumulation of cells in G1 was irreversible; removal of steroid in the presence of colcemid did not result in a decreased fraction of G1 cells. Furthermore, dexamethasone treatment did not protect cells against the effects of 33258 Hoechst-amplified killing of bromodeoxyuridine substituted cells exposed to light. These results show that dexamethasone arrests these leukemic cells in G1 and strongly suggest that dexamethasone-treated cells are killed upon entry into G1.     

Regulation of Cyclic AMP Formation in Cultures of Human Foetal Astrocytes by β2-Adrenergic and Adenosine Receptors

Two cell cultures, NEP2 and NEM2, isolated from human foetal brain have been maintained through several passages and found to express some properties of astrocytes. Both cell cultures contain adenylate cyclase stimulated by catecholamines with a potency order of isoprenaline greater than adrenaline greater than salbutamol much greater than noradrenaline, which is consistent with the presence of beta 2-adrenergic receptors. This study reports that the beta 2-adrenergic-selective antagonist ICI 118,551 is approximately 1,000 times more potent at inhibiting isoprenaline stimulation of cyclic AMP (cAMP) formation in both NEP2 and NEM2 than the beta 1-adrenergic-selective antagonist practolol. This observation confirms the presence of beta 2-adrenergic receptors in these cell cultures. The formation of cAMP in NEP2 is also stimulated by 5'-(N-ethylcarboxamido)adenosine (NECA) more potently than by either adenosine or N6-(L-phenylisopropyl)adenosine (L-PIA), which suggests that this foetal astrocyte expresses adenosine A2 receptors. Furthermore, L-PIA and NECA inhibit isoprenaline stimulation of cAMP formation, a result suggesting the presence of adenosine A1 receptors on NEP2. The presence of A1 receptors is confirmed by the observation that the A1-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine reverses the inhibition of isoprenaline stimulation of cAMP formation by L-PIA and NECA. Additional evidence that NEP2 expresses adenosine receptors linked to the adenylate cyclase-inhibitory GTP-binding protein is provided by the finding that pretreatment of these cells with pertussis toxin reverses the adenosine inhibition of cAMP formation stimulated by either isoprenaline or forskolin.     

Dexamethasone increases expression of mannose receptors and decreases extracellular lysosomal enzyme accumulation in macrophages

Macrophages express a mannose-specific pinocytosis receptor that binds and internalizes lysosomal hydrolases. Treatment of rat bone marrow-derived macrophages with dexamethasone resulted in a concentration- and time-dependent increase in mannose-receptor activity. The dexamethasone effect was maximal at 24 h. Half-maximal effects were observed at a dexamethasone concentration of 2.5 X 10(-9) M. With 125I-beta-glucuronidase as ligand, a 2.5-fold increase in uptake rate was observed in dexamethasone-treated cells, with no change in Kuptake (2.5 X 10(-7) M beta-glucuronidase). Cell surface binding (4 degrees C) was elevated 2.6-fold following dexamethasone treatment. The increase in ligand binding appeared to be due to an increase in number of sites with no change in affinity. Cycloheximide suppressed the dexamethasone-mediated rise in receptor number, while cycloheximide alone had little effect on receptor activity over 16 h. These results suggest that dexamethasone stimulates synthesis of mannose receptors in macrophages. Extracellular accumulation of hexosaminidase was sharply reduced by dexamethasone treatment, and corresponded with the rise in mannose-receptor activity. Extracellular levels of hexosaminidase from untreated macrophages were modestly increased by the presence of mannan, while the extracellular activity from dexamethasone-treated cells was increased significantly by mannan. Extracellular hexosaminidase, released from zymosan-treated macrophages, was dramatically reduced by dexamethasone pretreatment. Enzyme released from zymosan-stimulated macrophages was efficiently endocytosed by dexamethasone-treated cells in co-culture experiments, and this endocytosis was blocked by the addition of mannan. These results suggest that the mannose receptor of macrophages may play a role in regulating extracellular levels of lysosomal enzymes via a secretion-recapture mechanism.     

Specific [3H]SCH23390 Binding to Dopamine D1 Receptors in Cerebral Cortex and Neostriatum: Evidence for Heterogeneities in Affinity States and Cortical Distribution

The tritiated antagonist SCH23390 was used to identify dopamine D1 receptors in the cerebral cortex and neostriatum. The kinetic properties of binding were investigated in parallel experiments with membrane preparations from both tissues. The densities of receptors (Bmax) and the dissociation constants (KD) were determined from saturation curves, and the specificity of binding verified in competition experiments using agonists and antagonists. The cortical D1 receptor displays the same pharmacological selectivity (including stereospecificity) and kinetic properties as the neostriatal D1 receptor. From both the dissociation kinetics by dilution and the competition curves, it could be established that there is an heterogeneity of binding probably due to high- and low-affinity states. Endogenous dopamine, 4-hydroxy-3-methoxyphenylacetic acid, 3,4-dihydroxyphenylacetic acid, and 3-methoxytyramine contents, as well as D1 receptor distribution, were measured for the neostriatum and four localized cortical areas: anterior cingulate, primary somatosensory, primary visual, and piriform-entorhinal. For the regions examined, the distribution of D1 receptors is heterogeneous, but correlates very well (r greater than 0.98) with the endogenous levels of dopamine and its major metabolites.     

Autocrine activation of adenosine A1 receptors blocks D1A but not D1B dopamine receptor desensitization

Adenosine is known to modulate dopamine responses in several brain areas. Here, we show that tonic activation of adenosine receptors is able to impede desensitization of D1 dopamine receptors. As measured by cAMP accumulation in transfected COS-7 cells, long-term exposure to dopamine agonists promoted desensitization of D1B receptor but not that of D1A receptor. The inability of D1A receptor to desensitize was a result of the adenosine present in culture medium acting through activation of adenosine A1 receptors. Cell incubation with either adenosine deaminase, CGS-15943, a generic adenosine receptor antagonist, or the A1 antagonist DPCPX restored the long-term desensitization time-course of D1A receptors. In Ltk cells stably expressing A1 adenosine receptors and D1A dopamine receptors, pre-treatment of cells with R(-)-PIA, a full A1 receptor agonist, did not significantly inhibit the acute increase in cAMP levels induced by D1 receptor agonists, but blocked desensitization of D1A receptors. However, simultaneous activation of A1 and D1A receptors promoted a delayed D1A receptor desensitization. This suggests that functional interaction between A1 and D1A receptors may depend on the activation kinetics of components regulating D1 receptor responses, acting differentially on D1A and D1B receptors.     

Correlation in inflammatory fibroblasts between the number of glucocorticoid receptors and PGE2 release

The anti-inflammatory effects of glucocorticoids are mediated through steroid receptor occupancy and there is a significant correlation between the extent of receptor saturation and the extent of the biological effects. In a previously published study, we found that the number of these receptors was higher in inflammatory fibroblasts than in quiescent ones. PGE2 release, measured at the same time as the number of steroid receptors, was higher when the cells were from inflammatory tissue. Our aim, in the present study, was to determine whether the PGE2 released by cells during inflammatory processes could participate in increasing the number of steroid receptors. Fibroblasts obtained from rat quiescent subcutaneous connective tissue and granulomas were subcultured in monolayers. The specific binding of [3H]dexamethasone was assessed and analyzed by a method described by Kalimi et al. After a freeze-thaw cycle, we observed a decrease in the number of receptors in inflammatory fibroblasts. When the frozen and thawed fibroblasts were subcultured in the presence of PGE2 (10(-8) M), the number of receptors was enhanced in fibroblasts from inflammatory tissue. Cycloheximide (3 X 10(-7) M) prevented this increase. The release of PGE2 decreased after freezing and then increased simultaneously with the number of receptors in inflammatory cells. These findings suggest that PGE2 may play a role in regulating steroid effects on fibroblast function.     

Glucocorticoids increase cholecystokinin receptors and amylase secretion in pancreatic acinar AR42J cells

We recently reported in AR42J pancreatic acinar cells that glucocorticoids increased the synthesis, cell content, and mRNA levels for amylase (Logsdon, C.D., Moessner, A., Williams, J.A., and Goldfine, I.D. (1985) J. Cell Biol. 100, 1200-1208). In addition, in these cells glucocorticoids increased the volume density of secretory granules and rough endoplasmic reticulum. In the present study we investigate the effects of glucocorticoids on the receptor binding and biological effects of cholecystokinin (CCK) on AR42J cells. Treatment with 10 nM dexamethasone for 48 h increased the specific binding of 125I-CCK. This increase in binding was time-dependent, with maximal effects occurring after 48 h, and dose-dependent, with a one-half maximal effect elicited by 1 nM dexamethasone. Other steroid analogs were also effective and their potencies paralleled their relative effectiveness as glucocorticoids. Analyses of competitive binding experiments conducted at 4 degrees C to minimize hormone internalization and degradation revealed the presence of a single class of CCK binding sites with a Kd of approximately 6 nM and indicated that dexamethasone treatment nearly tripled the number of CCK receptors/cell with little change in receptor affinity. Treatment with 10 nM dexamethasone increased both basal amylase secretion and the amylase released in response to CCK stimulation. In addition, dexamethasone increased the sensitivity of the cells to CCK. The glucocorticoid decreased the concentration of CCK required for one half-maximal stimulation of amylase secretion from 35 +/- 6 to 8 +/- 1 pM. These data indicate, therefore, that glucocorticoids induce an increase in the number of CCK receptors in AR42J cells, and this increase leads to enhanced sensitivity to CCK.     

Interleukin 1 and tumor necrosis factor potentiate angiotensin II- and calcium ionophore-stimulated prostaglandin E2 synthesis in rat renal mesangial cells

In resting mesangial cells, angiotensin II and the calcium ionophore A23187 stimulated prostaglandin E2 (PGE2) formation. After pretreatment with interleukin 1 beta (IL-1 beta) or tumor necrosis factor alpha (TNF alpha), which are themselves potent stimuli for PGE2 synthesis, mesangial cells displayed an amplified response to angiotensin II and A23187. The cytokine-induced effects occurred in a time- and dose-dependent manner and were attenuated by actinomycin D, cycloheximide and dexamethasone. IL-1 beta and TNF alpha treatment also increased the amount of arachidonic acid released after stimulation of cells with angiotensin II and A23187. In addition, IL-1 beta but not TNF alpha treatment augmented the formation of PGE2 from exogenous arachidonic acid by mesangial cells. Furthermore, the conversion of prostaglandin H2 to PGE2 was not changed by IL-1 beta and TNF alpha. These results suggest that IL-1 beta and TNF alpha exert a priming effect on PGE2 production in mesangial cells.