Biomedical subjects

W J Chiou

Publications and source records attributed to W J Chiou.

36 records · Page 2Linked to original sources

PubMed1997

Endothelin receptor antagonists: effect of serum albumin on potency and comparison of pharmacological characteristics.

Endothelins (ETs) are 21-amino acid peptides that bind to membrane receptors to initiate pathophysiological effects. Two types of ET receptors, ETA and ETB, have been identified. Various ET receptor antagonists are being developed as therapeutic agents. This report examines the effects of bovine serum albumin (BSA) on the potency of ET receptor antagonists and compares five ET receptor antagonists. Competition studies show that in the absence of BSA, A-127722 and L-749329 inhibited ET-1 binding to ETA receptor with the same IC50 value of 0.09 nM. Addition of increasing concentrations of BSA incrementally decreased the potency of the antagonists: in the presence of 5% BSA, the IC50 values increased to 4.3 and 820 nM, respectively. Similarly, addition of BSA decreased the potency of antagonists in inhibiting ET-1-stimulated phosphatidylinositol hydrolysis. These results suggest that serum albumin has profound effects on the potencies of ET receptor antagonists. FR139317, PD-156707, L-749329, Ro-47-0203 and A-127722 were then selected for direct comparison under identical experimental conditions with 0.2% BSA. The potency of antagonists was assessed by binding studies for the determination of IC50 and Ki values and by ET-1-stimulated phosphatidylinositol hydrolysis and arachidonic acid release for the determination of IC50 and pA2 values. All five antagonists inhibited ET binding and the biological effects exerted by ET in a competitive mode. The Ki values for A-127722, PD-156707, FR139317, Ro-47-0203 and L-749329 for the ETA receptor were 0.07, 0.38, 0.80, 3.67 and 33.6 nM, respectively. A similar hierarchy was revealed by the functional assays. Our results suggest that the rank order of potency of the antagonists is A-127722 > or = PD-156707 > or = FR139317 > Ro-47-0203 > L-749329.

Acetamides↗

PubMed1997

Endothelin receptor in benign prostatic hyperplastic cells. Binding and functional studies.

Endothelins (ETs) are 21-amino acid peptides that bind to membrane receptors to initiate pathophysiological effects. This report characterizes ET receptors in benign prostatic hyperplasia-1 (BPH-1) cells, a prostate cell line isolated from a specimen of a 60-yr-old man with benign prostatic hyperplasia. [(125)I]ET-1 or -3 binding was of high affinity, with B(max) and K(d) values of 48 fmol/1 x 10(6) cells and 0.16 nM for ET-1, and 2.9 fmol/1 x 10(6) cells and 0.033 nM for ET-3, respectively. ET-1, ET-3, FR139317, Ro 46-2005, and IRL1620 inhibited [(125)I]ET-1 binding to these cells with IC50 values of 0.22, 186, 0.20, 52.8, and 772.3 nM, respectively. Reverse transcription-polymerase chain reaction confirmed that BPH-1 cells expressed more ET(A) than ET(B) receptors. ET-1 did not have any effect on arachidonic acid release, but caused a modest stimulation of phosphatidylinositol hydrolysis, and induced a prominent, sustained elevation in intracellular Ca2+ concentrations. The functional effects of ET-1 were completely inhibited by the ET(A)-selective antagonists FR139317 and A-127722, suggesting that the effects were mediated by the ET(A) receptor. These results suggest that ET may play functional roles in benign prostatic hyperplasia.

Arachidonic Acid↗

PubMed1996

2,4-Diarylpyrrolidine-3-carboxylic acids--potent ETA selective endothelin receptor antagonists. 1. Discovery of A-127722.

We have discovered a novel class of endothelin (ET) receptor antagonists through pharmacophore analysis of the existing non-peptide ET antagonists. On the basis of this analysis, we determined that a pyrrolidine ring might replace the indian ring in SB 209670. The resultant compounds were readily prepared and amenable to extensive SAR studies. Thus a series of N-substituted trans,trans-2-(4-methoxyphenyl)-4-(1,3-benzodioxol-5-yl)pyrroli din e-3- carboxylic acids (8) have been synthesized and evaluated for binding at ET(A) and ET(B) receptors. Compounds with N-acyl and simple N-alkyl substituents had weak activity. Compounds with N-alkyl substituents containing ethers, sulfoxides, or sulfones showed increased activity. Much improved activity resulted from compounds where the N-substituents were acetamides. Compound 17u (A-127722) with the N,N-dibutylacetamide substituent is the best of the series. It has an IC(50)=0.36 nM for inhibition of ET-1 radioligand binding at the ET(A) receptor, with a 1000-fold selectivity for the ET(A) vs the ET(B) receptor. It is also a potent inhibitor (IC(50)=0.16 nM) of phosphoinositol hydrolysis stimulated by ET-1, and it antagonized the ET-1-induced contraction of the rabbit aorta with a pA(2)=9.20. The compound has 70% oral bioavailability in rats.

Animals↗

PubMed1996

Endothelins and endothelin receptor antagonists: binding to plasma proteins.

Endothelins (ET) are 21-amino acid peptides that bind to membrane receptors to initiate a wide range of pathophysiological effects. PD-156707, L-749329, Ro-470203, and A-127722 are potent non-peptide ET receptor antagonists developed recently. When tested in human and rat plasma, both ET-1 and -3 and the four aforementioned antagonists exhibited a high degree (> 98%) of plasma protein binding. When ET-1 binding to the receptors was examined, 5% (v/v) of human plasma inhibited ET-1 binding to both ETA and ETB receptors by 80 - 90%. Similarly, 5% (w/v) of human serum albumin inhibited ET-1 binding by 82%, suggesting that the major protein component in plasma which interfered with ET-1 binding to the receptors was serum albumin. Competition studies show that, in the absence of human serum albumin, the IC50 values of PD-156707, L-749329, Ro-47-0203, and A-127722 were 0.37, 0.29, 5.7, and 0.22 nM, respectively. Addition of increasing doses of human serum albumin incrementally decreased the potency of the antagonists; in the presence of 5% of human serum albumin, the IC50 values increased to 62.8, 50.2, 122.7, and 6.72 nM for PD-156707, L-749329, Ro-47-0203, and A-127722, respectively. In conclusion, ET and ET receptor antagonists exhibit a high degree of binding to plasma proteins, especially serum albumin. Consequently, serum albumin inhibits ET binding to its receptors, and also decreases the potency of ET receptor antagonists. Our findings may explain the discrepancy observed for ET receptor antagonists between in vitro and in vivo potencies.

3T3 Cells↗

PubMed1996

Pharmacological characterization of A-127722: an orally active and highly potent ETA-selective receptor antagonist.

Endothelins (ET) are potent vasoactive peptides implicated in the pathogenesis of a number of vascular diseases. The effects of ET on mammalian organs and cells are initiated by binding to ETA or ETB receptors. In this report, we document the pharmacology of A-127722, a novel ETA-selective receptor antagonist. A-127722 inhibits [125I]ET-1 binding to cloned human ETA and ETB receptors competitively with Ki values of 69 pM and 139 nM, respectively. A-127722 exhibits a dose-dependent inhibition of ET-1-induced arachidonic acid release in human pericardium smooth muscle cells with a pA2 value of 10.5 and inhibits ET-1-induced vasoconstriction in isolated rat aorta with a pA2 value of 9.2. In vivo, A-127722 dose-dependently blocks the pressor response to ET-1 (0.3 nmol/kg i.v.) in conscious rats. Statistically significant (P < .05) antagonism is seen at doses greater than 0.1 mg/kg p.o. Maximal inhibition, at 10 mg/kg, remains constant for at least 8 hr after dosing. No effect is seen on the ETB-mediated transient vasodepressor effect of exogenous ET-1. In conclusion, A-127722 is ETA-selective, orally bioavailable and efficacious for inhibiting the effects of ET in the rat, and A-127722 is the most potent ET receptor antagonist yet reported.

Administration, Oral↗

PubMed1995

Oxidant-induced activation of protein kinase C in UC11MG cells.

Free radical formation and subsequent lipid peroxidation may participate in the pathogenesis of tissue injury, including the brain injury induced by hypoxia or trauma and cardiac injury arising from ischemia and reperfusion. However, the exact cellular mechanisms by which the initial oxidative insult leads to the ultimate tissue damage are not known. A number of reports have indicated that protein kinase C (PKC) may be activated following oxidative stress and that this enzyme may play an important role in the steps leading to cellular damage. In this work, we have examined in a cell model whether PKC is activated following oxidative exposure. UC11MG cells, a human astrocytoma cell line, were treated with H2O2. Incubation with 0.5 mM H2O2 increased malondialdehyde levels by as early as 15 minutes. To assess the effects of H2O2 treatment on PKC activation, we measured phosphorylation of an endogenous PKC substrate, the MARCKS (myristoylated alanine-rich C kinase substrate) protein. Treatment of cells with 0.2-1.0 mM H2O2 resulted in a rapid increase in MARCKS phosphorylation. Phosphorylation was stimulated approximately 2.5-fold following treatment with 0.5 mM H2O2 for ten minutes. Treatment with phorbol 12-myristate 13-acetate, a PKC activator, increased MARCKS phosphorylation approximately 4-fold. The H2O2-induced MARCKS phosphorylation was inhibited by the addition of the kinase inhibitors H-7 and staurosporine. Furthermore, specific down-regulation of PKC by phorbol ester also inhibited H2O2-induced MARCKS phosphorylation. These results indicate that PKC is rapidly activated in cells following an oxidative exposure and that this cell system may be a good model to further investigate the role of PKC in regulating oxidative damage in the cell.

Down-Regulation↗

PubMed1995

Endothelin receptor in human astrocytoma U373MG cells: binding, dissociation, receptor internalization.

Endothelin (ET) receptor in human astrocytoma U373MG cells was characterized. ET-1, ET-3, sarafotoxin S6C, IRL1620, BQ788, Ro46-2005 and PD142893 inhibited specific [125I]ET-1 binding with Ki values of 0.03 0.06, 0.74, 5.01, 4.45, 2275 and 157 nM, respectively. ETA selective antagonists BQ123 and FR139317 at 1 microM did not block [125I]ET-1 binding. Reverse transcription-polymerase chain reaction confirmed the results from competition studies that U373 cells expressed predominantly ETB receptor. The Bmax and KD values of [125I]ET-1 binding were 0.15 pmol/1 x 10(6) cells and 0.23 nM. The molecular mass for the receptor was 45 kDa. ET-1 binding did not stimulate Ca+2 mobilization, phosphatidylinositol hydrolysis or arachidonic acid release, nor did it affect the intracellular cAMP or cGMP level. Interestingly, a majority of ET (> 80%) bound to the receptor was rapidly internalized, consistent with emerging evidence that a major function of ETB receptor is to clear ET. [125I]ET-1 binding was time-dependent and bound [125I]ET-1 was difficult to dissociate. In contrast, bound antagonists were much easier to dissociate. The results suggest that agonists and antagonists of the ET receptor exhibited different dissociation characteristics, with antagonist binding more reversible than agonist binding.

Astrocytoma↗

PubMed1995

Dissociation characteristics of endothelin ETA receptor agonists and antagonists.

Endothelin (ET) binding to receptors has been shown to be almost irreversible. We studied the dissociation characteristics of ETA receptor agonists and antagonists using membranes prepared from rat pituitary cells (MMQ). Consistent with our previous report, competition studies comparing ET-1, ET-3, BQ123, FR139317, PD142893, and Ro46-2005 show that MMQ cells contained predominantly the ETA receptor. [125I]ET-1 bound to the receptor was difficult to dissociate. In contrast, bound BQ123, FR139317, and Ro46-2005 were easier to dissociate, suggesting that antagonist binding was more reversible. Although BQ123 (5 nM), FR139317 (1 nM), and Ro46-2005 (0.5 microM) inhibited 0.1 nM [125I]ET-1 binding by greater than 80% after 15 min of incubation, the inhibition decreased to less than 20% after 24 h of incubation. This decrease in binding inhibition was not the result of antagonist degradation. These results suggest that, similar to our previous observation made with the ETB receptor in porcine cerebellum, the ability of antagonists to inhibit [125I]ET-1 binding to the ETA receptor is critically dependent on the incubation time because of the difference in the dissociation characteristics between antagonists and ET-1.

Amino Acid Sequence↗

PubMed1994

Endothelin receptor agonists and antagonists exhibit different dissociation characteristics.

Endothelins (ETs) are vasoconstricting peptides that bind to membrane receptors to initiate their physiological effects. This report compares the dissociation characteristics of selected ET agonists and antagonists, and studies the effects of any difference in dissociation characteristics on the potency of antagonists. Competition studies using various ET receptor ligands against [125I]ET-1 or [125I]ET-3 binding demonstrated that porcine cerebellum membranes contain predominantly ETB receptor. [125I]IRL1620 associated with the receptors in a time-dependent manner. Although bound [125I]IRL1620 was easier to dissociate than bound [125I]ET-3, both agonists exhibited a dissociation half life > 20 h. For non-radiolabeled ligands, bind-and-wash studies were employed in which membranes were pre-incubated with unlabeled ligand followed by extensive washing before assaying for [125I]ET-1 binding. Results from bind-and-wash studies confirmed that bound non-radiolabeled IRL1620 and ET were as difficult to dissociate as [125I]ligands. In contrast, bound PD142893 and Ro46-2005 were easily dissociated from ETB receptors. Consequently, the inhibitory effects of PD142893 and Ro46-2005 on [125I]agonist binding diminished following incubation time. In cloned human ETA and ETB receptors, bound ET-1 was also more difficult to dissociate than bound antagonists. These results suggest that the differences in the dissociation characteristics of ET receptor agonists vs. antagonists may account for the diminished potency of Ro46-2005 and PD142893 as a function of incubation time.

Amino Acid Sequence↗

PubMed1994

Endothelin receptor antagonists exhibit diminishing potency following incubation with agonist.

Endothelins (ET) are 21-amino acid peptides that bind to membrane receptors to initiate a wide range of pathophysiological effects. ET binding to receptors has been shown to be almost irreversible because bound ET is difficult to dissociate. This report studies the dissociation characteristics of receptor antagonists and further examines the effects of ET's difficult-to-dissociate binding on the potency of antagonists. In membranes prepared from porcine cerebellum, [125I]ET-1 binding was effectively blocked by ET-1 and ET-3 with similar IC50 values (0.08 nM vs. 0.17 nM), suggesting that porcine cerebellum contains predominantly the ETB receptor subtype. [125I]ET-3 binding was inhibited by Ro46-2005 and PD142893, two non-selective antagonists, with IC50 values of 570 +/- 50 nM and 410 +/- 100 nM, respectively. Consistent with previous observations, bound [125I]ET-1 in porcine cerebellum membranes was also difficult to dissociate. In contrast, bound Ro46-2005 or PD142893, but not bound ET-1, could be readily washed away from membranes, suggesting that antagonist binding was more reversible than ET-1 binding. Although Ro46-2005 or PD142893 at 0.5 microM inhibited 0.1 nM [125I]ET-1 binding by > 80% after 15 min of incubation, the inhibitory effect decreased to approximately 50% after 3 h of incubation, and further decreased to < 10% at 24 h. This decrease in antagonizing potency was further confirmed by the results that the IC50 values of the two antagonists against [125I]ET-3 binding increased with increasing incubation time. Control experiments indicate that the observed decrease in the potency of Ro46-2005 and PD142893 was not the result of ligand degradation. These results suggest that the potency of antagonists is critically dependent on the incubation time because antagonist binding is more reversible than ET binding.

Amino Acid Sequence↗

PubMed1994

Characterization of two endothelin converting enzymes and their preference for big endothelin-1 and -2 as substrates.

Two proteolytic activities that convert big ET to ET at neutral pH were identified in solubilized membranes prepared from rat lung. The endothelin-converting activities were partially purified by using A80227 ((2S,3R,4S)-2-([N-acetylcyclohexylalanyl-isoleucyl]amino)-1-(2-nap hthyl)-3,4-dihydroxy-6-methylheptane) coupled to an affinity-gel column (Affigel), and subsequently by concanavalin-A immobilized gel chromatography. An endothelin-converting activity was identified in the fraction containing proteins that did not bind to A80227-Affigel. This protease was sensitive to phosphoramidon, soybean trypsin inhibitor, and chymostatin, and preferred big ET-1 or big ET-2 as its substrate over bit ET-3. A second endothelin-converting activity was identified in the fraction containing proteins that bound to the A80227-coupled gel and was eluted by raising the pH. This protease exhibited activities throughout a range of pH 5.5-9.5, was inhibited by pepstatin A and A80227, and also preferred big ET-1 or big ET-2 over big ET-3 as its substrate. Both enzymes were glycoproteins based on their binding to concanavalin-A immobilized gel and were readily eluted by a buffer containing 0.5 M manopyranoside. The results from the pH and protease inhibitor profiles suggesting that these two ET-converting activities extracted from rat lung membranes are distinct and are different from the previously reported endothelin-converting enzymes.

Animals↗

PubMed1994

Endothelin receptors in human smooth muscle cells: antagonist potency differs on agonist-evoked responses.

The present study characterized endothelin (ET) receptors in human pericardium smooth muscle cells (HPSMC) and examined the potency of antagonists on ET-evoked signal transduction and DNA synthesis. HPSMC contain both ETA and ETB receptors. ET-1 binding was completely inhibited by a nonselective antagonist (Ro-46-2005) or a combination of ETA-selective and ETB-selective ligands (BQ-123 and ET-3). The molecular masses for ETA and ETB receptors were 69 and 42 kDa, respectively. ET-1, but not ET-3, stimulated phosphatidylinositol hydrolysis and arachidonic acid release in a dose- and time-dependent manner, reaching a plateau within 20-40 min. These immediate effects of ET-1 on signal transduction were completely inhibited by 1 microM, BQ-123, ET-1, but not ET-3, stimulated DNA synthesis in a dose-dependent manner, and the effect became prominent after 24 h. BQ-123 (1 microM) or Ro-46-2005 (10 microM) did not completely inhibit this mitogenic effect of ET-1. The reduced potency of BQ-123 and Ro-46-2005 on the delayed effect of ET-1 was not the result of ligand degradation or a difference in receptor internalization; rather, the decrease in potency was due to the fact that antagonist binding was more reversible than ET-1 binding.

Binding Sites↗

PubMed1993

Prolonged treatment by phosphoramidon modulates the number of endothelin receptors in cultured Swiss 3T3 fibroblasts.

Endothelin (ET) is generated from prepro-ET initially by dibasic pair proteolysis, followed by a specific proteolytic cleavage between Trp21 and Val22. Currently, intense research is focused on the investigation of a metalloprotease that is inhibited by phosphoramidon (PHOS) only, but not by other protease inhibitors. In this report, we show that ET binding was increased significantly in cultured Swiss 3T3 fibroblasts with PHOS pretreatment. The effect of PHOS was dose- and time-dependent. Other protease inhibitors, such as thiorphan, pepstatin-A, E-64, phenylmethyl sulfonyl fluoride, and aprotinin, failed to exert a similar effect. Control experiments indicated that the effect of PHOS was not due to inhibition of 125I-ET-1 degradation. Binding studies using whole cells or membranes prepared from cell show that ET binding sites increased from 23,000 to 133,000 sites/cell in control versus PHOS-treated cells. The effect of PHOS treatment on the ET receptor may be due to the inhibition of a protease responsible for ET-receptor processing. This effect is likely to complicate interpretation of results from studies using PHOS to block the putative ET-converting enzyme.

3T3 Cells↗

PubMed1993

Phosphoramidon modulates the number of endothelin receptors in cultured Swiss 3T3 fibroblasts.

Endothelin (ET) is generated from prepro-ET by dibasic pair proteolysis, followed by specific proteolytic cleavage between Trp21 and Val22. Currently, intense research efforts are focused on the investigation of a metalloprotease-like ET-converting enzyme that is inhibited by phosphoramidon but not by other inhibitors of neutral metalloproteases. In this report, we show that ET binding was increased significantly in cultured Swiss 3T3 fibroblasts after phosphoramidon treatment. Saturation studies using membranes prepared from cells or using intact cells assayed at 4 degrees showed that Bmax increased from 0.13 pmol/mg or 0.038 pmol/1 x 10(6) cells in untreated cells to 0.66 pmol/mg or 0.22 pmol/1 x 10(6) cells in cells treated with 100 microM phosphoramidon for 24 hr, equivalent to a net increase of 100,000 ET binding sites/cell. The effect of phosphoramidon was time and dose dependent. Other protease inhibitors, such as thiorphan, pepstatin A, E-64, phenylmethylsulfonyl fluoride, bestatin, and leupeptin, failed to exert a similar effect. Reverse phase high performance liquid chromatography analysis indicated that the effect of phosphoramidon was not due to inhibition of 125I-ET-1 degradation. The effect of phosphoramidon remained evident after cells were treated with actinomycin D or cycloheximide to inhibit protein synthesis, suggesting that the phenomenon was not due to the effect of phosphoramidon stimulating the synthesis of ET receptors. Degradation studies suggested that the effect of phosphoramidon was due to inhibition of a protease responsible for degrading the ET receptor. The fact that Swiss 3T3 cells treated with phosphoramidon exhibit an increase in the number of ET receptors is likely to complicate the interpretation of results when phosphoramidon or related compounds are used to block the putative ET-converting enzyme.

3T3 Cells↗

PubMed1990

Two signal transduction pathways mediate interleukin-1 receptor expression in Balb/c3T3 fibroblasts.

Our previous studies showed that platelet-derived growth factor (PDGF) modulated interleukin-1 (IL-1) activity and IL-1 binding to Balb/c3T3 fibroblasts (Bonin, P. D., and Singh, J. P. (1988) J. Biol. Chem. 263, 11052-11055). Subsequent studies have demonstrated an action of PDGF at the level of IL-1 receptor (IL-1R) gene expression. PDGF treatment of Balb/c3T3 cells produces a 10-20-fold stimulation of mRNA for IL-1 receptor. Investigation of the signal transduction pathways shows that activation of either the protein kinase C pathway or the cAMP-mediated pathway leads to the stimulation of IL-1 receptor expression in Balb/c3T3 cells. Treatment of Balb/c3T3 cells with phorbol 12-myristate 13-acetate (PMA), a known activator of protein kinase C, produced an increased 125I-IL-1 binding to cells and stimulation of IL-1R mRNA. Staurosporine, an inhibitor of protein kinase C, blocked the induction of IL-1 binding by PDGF or PMA. Down-regulation of protein kinase C by pretreatment with PMA reduced the subsequent stimulation by PDGF. Chronic treatment with PMA, however, did not produce a complete inhibition of PDGF effect on IL-1R. Further studies showed that the agents that stimulate cAMP accumulation (isobutyl methylxanthine, dibutyryl), directly stimulate adenylate cyclase (forskolin), or activate G protein (choleragen) stimulated 125I-IL-1 binding and IL-1R mRNA accumulation in Balb/c3T3 cells. These studies suggest that potentially two signal transduction pathways mediate IL-1 receptor expression in Balb/c3T3 fibroblasts. Evidence is presented that suggests that stimulation of IL-1R through these two pathways (PMA/PDGF-stimulated and cAMP-stimulated) occurs independent of each other.

Alkaloids↗

PubMed1990

Obligatory action of polypeptide growth factors for the IL-1-mediated prostaglandin E2 production in fibroblasts. Potential role of growth factors in modulation of tissue response to IL-1.

Our studies show that in connective tissue cells, induction of PGE2 synthesis in response to IL-1 requires costimulation with platelet-derived growth factor (PDGF) or fibroblast growth factor (FGF). In cells incubated in medium containing fresh serum, IL-1 induced a dose-dependent synthesis of PGE2. However, when the cells were incubated in medium containing low serum or platelet poor plasma (lacking PDGF), IL-1 alone failed to induce PGE2 synthesis. PGE2 synthesis was restored when platelet poor plasma was supplemented with PDGF. Addition of PDGF or FGF together with IL-1 resulted in a 14- and 66-fold stimulation of PGE2 synthesis, respectively. Stimulation was dependent on the concentration of both IL-1 and the growth factor. PGE2 synthesis was also dependent on the synthesis of new proteins. In cells simultaneously treated with IL-1 and PDGF, PGE2 synthesis was initiated after a lag of 2 to 3 h, proceeded first with a rapid rate for 6 h, and then with a slower rate through 24 h. PGE2 synthesis during the latter, slower phase was greatly enhanced by pretreatment with PDGF, but not by pretreatment with IL-1. PDGF pretreatment also resulted in maintenance of 10- to 12-fold higher cell surface IL-1-binding during this phase. These data provide evidence for potentially novel interactions between PDGF and IL-1 activities, one of which is the modulation of IL-1 receptors by PDGF. Furthermore, these studies suggest that by virtue of their effect on IL-1 activities, PDGF and FGF may play additional roles in connective tissues, including an indirect role in inflammatory processes.

Animals↗

PubMed1989

Platelet-derived growth factor induces interleukin-1 receptor gene expression in Balb/c 3T3 fibroblasts.

Our previous studies have demonstrated that platelet-derived growth factor (PDGF) modulated cellular responses to interleukin-1 (IL-1). In this communication, we show that PDGF regulates expression of IL-1 receptor (IL-1 R) gene. Treatment of quiescent cultures of Balb/c 3T3 fibroblasts with PDGF produced 20-30-fold stimulation of IL-1 R mRNA with a concomitant increase in cell surface 125I-binding. IL-1 R mRNA accumulation occurred after an initial lag period and with a time course preceding the increase in 125I-IL-1 binding to cells. Induction of IL-1 R mRNA was blocked by inhibitors of protein synthesis, suggesting that a product of a gene expressed immediately after PDGF addition is required for IL-1 R gene expression. These latter data provide evidence for an ordered sequence of expression of PDGF-inducible "immediate early" gene(s) and IL-1 R gene. These results suggest that in connective tissues, PDGF may be an important determinant in initiating and maintaining cellular responses to IL-1. Such responses may have important consequences in the actions of IL-1 under normal and pathological conditions such as arthritis and atherosclerosis.

Animals↗

PubMed1987

A possible role for dephosphorylation in glucocorticoid receptor transformation.

Addition of bovine intestinal alkaline phosphatase to mouse AtT-20 cell cytosol increases the rate of glucocorticoid receptor transformation, as evidenced by a change in sedimentation rate from 9.1S to 5.2S. Acid phosphatases are completely ineffective in this regard. Alkaline phosphatase-promoted receptor transformation is both time- and dose-dependent. A variety of phosphatase inhibitors are effective in inhibiting this process, the most potent being transition metal oxyanions such as molybdate, tungstate, and arsenate. The ability of the various inhibitors to suppress alkaline phosphatase-promoted receptor transformation does not correspond well with their potencies for inhibiting para-nitrophenyl phosphate hydrolysis. However, a better correspondence between the inhibition of endogenous receptor transformation and total cytosolic phosphatase activity is observed, and both sodium fluoride and glucose-1-phosphate inhibit endogenous receptor transformation. The protease inhibitors phenyl-methylsulfonyl fluoride and antipain have no effect on receptor transformation. Surprisingly, leupeptin is effective in inhibiting alkaline phosphatase-promoted receptor transformation. Although this raises the possibility of a contaminating protease activity in the alkaline phosphatase enzyme preparation, treatment of covalently affinity-labeled receptor with the enzyme shows no proteolysis of the receptor or any other non-specifically labeled cytosolic protein. Thus, it is possible that a novel action of leupeptin, unrelated to its protease-inhibitory activity, may be involved in the suppression of receptor transformation. The studies presented here suggest that dephosphorylation of some component in cytosol is involved in the destabilization of receptor subunit interactions, resulting in glucocorticoid receptor transformation.

Alkaline Phosphatase↗