Ligand-induced expression of peroxisome proliferator-activated receptor alpha and activation of fatty acid oxidation enzymes in fatty liver.

BACKGROUND: Peroxisome proliferator-activated receptor alpha (PPARalpha) regulates lipid metabolism upon activation by ligands. Peroxisome proliferator-activated receptor alpha may play a role in the pathogenesis of fatty liver disease. The aim of this study was to assess the PPARalpha expression pattern and mitochondrial/peroxisomal enzyme activities in response to high fat diet (HFD) and clofibrate, a well known PPARalpha ligand. MATERIALS AND METHODS: Four groups of Wistar-Albino rats were included: (1) rats fed a control diet (CD) for 6 weeks, (2) rats fed CD (6 weeks) plus clofibrate (last 2 weeks), (3) rats fed HFD for 6 weeks, and (4) rats fed HFD (6 weeks) plus clofibrate (last 2 weeks). Peroxisome proliferator-activated receptor alpha expression was evaluated by immunohistochemistry. Fatty acid beta-oxidation (peroxisomal-acyl-CoA-oxidase and mitochondrial-acyl-CoA-dehydrogenase) and catalase enzyme activities, and malondialdehyde and glutathion levels were measured spectrophotometrically in liver tissues. RESULTS: All animals were fed HFD but only 2/12 animals were fed HFD plus clofibrate-developed fatty liver. Both HFD and clofibrate induced PPARalpha expression, clofibrate induction being more prominent than HFD. Clofibrate plus HFD did not further increase PPARalpha expression. Activities of peroxisomal-acyl-CoA-oxidase and mitochondrial-acyl-CoA-dehydrogenase enzymes were not induced by HFD alone. Clofibrate increased the activity of these enzymes in both CD- and HFD-fed animals. However, an increase of acyl-CoA-oxidase activity was blunted in rats fed HFD. Catalase activity and malondialdehyde levels were increased but glutathion levels were unchanged in rats fed HFD plus clofibrate. CONCLUSIONS: Clofibrate was a more potent inducer of PPARalpha expression than HFD in our rat fatty liver model. The finding of blunted peroxisomal enzyme response to clofibrate in fatty livers suggests that alterations in postreceptor events may exist and further contribute to liver steatosis. Clofibrate seems to stabilize glutathion content and this might contribute to the prevention of liver steatosis.

Reporter cell lines are useful tools for monitoring biological activity of nuclear receptor ligands.

To characterize the specificity of synthetic compounds for nuclear receptors, we established stable cell lines expressing the luciferase gene and different wild-type or chimaeric receptors. MCF-7 cells, which express the oestrogen receptor alpha (ER alpha), and HeLa cells, which do not express the oestrogen receptor, were transfected with a plasmid containing the luciferase gene downstream from a minimum promoter (beta-globin) and an oestrogen-responsive element, generating the MELN and the HELN cell lines, respectively. MELN cells enabled the detection of compounds that bind to the ER alpha or interfere with its pathway. HELN cells were used to establish stable transfectants expressing different nuclear receptors containing the DNA-binding domain of the oestrogen receptors. We thus established ER alpha or ER beta reporter cell lines by transfecting ER alpha or ER beta expression plasmids, and also retinoic acid receptor alpha, beta or gamma reporter cell lines by transfecting the chimaeric RAR gene, in which the DNA-binding domain was replaced by the ER alpha DNA-binding domain.

In vitro and in vivo inhibition of myeloperoxidase with 5-fluorouracil.

OBJECTIVE: Myeloperoxidase (MPO) exists in neutrophils and has an important bactericidal role. During phagocytosis, MPO catalyzes a peroxidative reaction using chloride ion and hydrogen peroxide (H2O2) as substrate. The aim of the present study was to investigate whether 5-fluorouracil (5-FU), a chemotherapeutic agent, has a direct inhibitory effect on MPO and to evaluate some properties of this inhibition. METHODS: The inhibitory effect of 5-FU on MPO was studied in rat tissue, human leukocytes, and leukocytes from cancer patients under 5-FU therapy. MPO was solubilized in a detergent-containing buffer. MPO activity was measured spectrophotometrically through the oxidation of a synthetic substrate tetramethyl benzidine in the presence of H2O2. RESULTS: 5-FU inhibited tissue-associated MPO activity in a dose-dependent but not time-dependent manner with an IC50 value of 0.6 mg/ml. 5-FU also inhibited MPO activity in isolated human leukocytes in a dose-dependent manner, and the IC50 value was 0.75 mg/ml. Using this 5-FU concentration, the inhibitory effect was monitored at different substrate concentrations. Leukocyte MPO activities of patients receiving 5-FU therapy were compared before treatment and after the first, second, and third administration cycles. 5-FU treatment resulted in a significant decrease in leukocyte MPO activity, and repeated 5-FU treatment caused additional decrease. CONCLUSION: Our data showed that 5-FU directly inhibited the MPO activity of human leukocytes in vitro and in vivo. We concluded that, the patients treated with 5-FU should be intensively followed for the risk of infections.

Role of nitric oxide in hypoxia-induced changes in newborn rats.

In order to investigate the role of nitric oxide (NO) in hypoxic tissue damage in newborns, we studied the effects of systemic administration of an inhibitor of NO synthase, N(G)-nitro-L-arginine (L-NNA), and the precursor for the synthesis of NO, L-arginine (L-ARG), on the biochemical and histological changes in brain, heart, lung, liver, kidney, intestine, and skeletal muscle tissues. Four groups of 1-day-old Wistar rat pups were used: control, hypoxic, L-ARG, and L-NNA groups. L-ARG 100 mg/kg or L-NNA 2 mg/kg was administered as a bolus intraperitoneally 1.5 h before hypoxia. Hypoxia increased lipid peroxidation in all tissues except muscle; this increase was prevented by L-NNA and L-ARG in brain, heart, lung, kidney, and liver tissues. L-NNA in intestine and L-ARG in muscle tissue increased lipid peroxidation. The tissue-associated myeloperoxidase activity was decreased in the liver by L-NNA and L-ARG. Histopathological changes in intestines were villous epithelial separation and hyperemia in hypoxic and L-NNA groups which were not observed in control and L-ARG groups. In lungs, pulmonary hemorrhage was observed only in the hypoxic group. These data suggest that NO acts both as a destructive and a protective agent in the pathogenesis of hypoxia-reoxygenation injuries.

Comparison of the effects of melatonin and methylprednisolone in experimental spinal cord injury.

OBJECT: Melatonin is a very effective antioxidant agent. This study was performed to investigate the effects of melatonin in experimental spinal cord injury (SCI). The authors also compared its effects with those of methylprednisolone, which also protects the spinal cord from secondary injury because of its antioxidant effect on membrane lipids. METHODS: Adult male albino rats were used for the study, and paraplegia was produced using a previously described weight-drop technique. Melatonin and methylprednisolone were given intraperitoneally by bolus injections of 100 mg/kg and 30 mg/kg, respectively, immediately after induction of trauma. The animals were killed, and 1-cm samples of injured spinal cord were obtained at 1, 24, and 48 hours postinjury. Lipid peroxidation was estimated by thiobarbituric acid test. Electron microscopic studies were performed to determine the effects of melatonin on neurons, axons, and subcellular organelles after experimental SCI. A grading system was used for quantitative evaluation. Following SCI, there was significant increase in lipid peroxidation. In melatonin- and methylprednisolone-treated groups, lipid peroxidation was found to decrease to the baseline (preinjury) levels. There was a significant difference between trauma-alone and treatment groups, but no statistical difference was found between the melatonin- and methylprednisolone-treated groups. Electron microscopic findings showed that SCI produced by the weight-drop technique resulted in profound tissue damage. CONCLUSIONS: Both melatonin and methylprednisolone have been shown to protect neuron, axon, myelin, and intracellular organelles including mitochondrion and nucleus. However, this study provides quantitative evidence that this protection of neurons and subcellular organelles of spinal cord after secondary injury is much more obvious in melatonin-treated rats than those treated with methylprednisolone. In view of these data, melatonin has been shown to be very effective in protecting the injured spinal cord from secondary injury.

Effects of intestinal ischemia-reperfusion on major conduit arteries.

Intestinal ischemia-reperfusion (I-R) is a common and serious clinical condition associated with simultaneous remote organ dysfunction. The purpose of this study was to investigate the effects of intestinal I-R on the vasomotor functions of major conduit arteries. Anesthetized rabbits were randomly assigned to one of three groups: sham-operated controls (Group I), and one-hour intestinal ischemia with two-hour reperfusion (Group II) or four-hour reperfusion (Group III). The following mechanisms of vasomotor functions were studied in abdominal aorta, superior mesenteric, renal, pulmonary, and carotid arterial rings: (1) endothelial-dependent vasodilation response to acetylcholine, (2) endothelial-independent vasodilation response to nitroprusside, (3) beta-adrenergic vasodilation response to isoproterenol, and (4) phenylephrine-induced vasoconstriction. Intestinal injury was quantified using malondialdehyde (MDA) concentration and wet-to-dry intestine weight ratio. Intestinal I-R did not affect the maximal responsiveness or the sensitivity to acetylcholine, nitroprusside, and isoproterenol in all the vessels studied. The maximal contractile response to phenylephrine increased significantly in mesenteric artery in Group II, (227.1+/-15.1% vs. 152.8+/-11.7% in controls) (p<0.05). Intestinal MDA concentration, a marker of oxidant injury, increased from 39.87+/-9.41 nmol/g to 67.8+/-8.8 nmol/g in group II (p<0.01), and to 94.8+/-7.56 nmol/g in Group III (p<0.001). Wet-to-dry intestine weight ratio increased from 3.62+/-0.12 to 4.28+/-0.17 in Group II (p<0.01), to 4.62+/-0.14 in Group III (p<0.001). These data indicate that although the intestines of the animals subjected to intestinal I-R are seriously injured, the smooth muscle relaxation of major conduit arteries was not affected.

Intestinal ischemia and reperfusion impairs vasomotor functions of pulmonary vascular bed.

OBJECTIVE: To investigate the effects of intestinal ischemia and reperfusion (I/R) on the pulmonary vascular endothelium and smooth muscle. SUMMARY BACKGROUND DATA: Respiratory failure is an important cause of death and complications after intestinal I/R. Although the mechanism of respiratory failure in this setting is complex and poorly understood, recent studies of lung injury suggest that endothelial dysfunction may play a significant role. METHODS: A rat model of acute lung injury was studied after 60 minutes of superior mesenteric arterial occlusion followed by either 120 or 240 minutes of reperfusion. The pulmonary vasomotor function was examined in isolated lungs perfused at a constant flow rate. RESULTS: Sixty minutes of intestinal ischemia followed by 120 or 240 minutes of reperfusion led to a significant reduction in the ability of the pulmonary vasculature to respond to angiotensin II, acetylcholine, and calcium ionophore but not to nitroglycerin. The vasoconstriction response to N(G)-nitro-L-arginine methyl ester, which is a measure of basal nitric oxide release, was diminished in the 240-minute reperfusion group. Intestinal I/R was also associated with pulmonary leukosequestration and increased pulmonary microvascular leakage. CONCLUSIONS: Basal and agonist-stimulated release of nitric oxide from the pulmonary vascular endothelium and the ability of pulmonary smooth muscle to contract in response to angiotensin II were impaired by intestinal I/R. Such functional impairment in both pulmonary vascular endothelium and smooth muscle may contribute to the alveolocapillary dysfunction and pulmonary hypertension found in acute lung injury after intestinal I/R.

Antioxidant action of the antiarrhythmic drug mexiletine in brain membranes.

Mexiletine is a class Ib antiarrhythmic drug used in the treatment of ventricular arrhythmias. The Na+ channel blocker mexiletine inhibits calcium influx in cells via decreasing reverse operation of the Na+-Ca2+ exchanger. Thus this drug is shown to protect the CNS white matter against anoxic/ischemic injury. The aim of our study was to investigate if this drug could act as an antioxidant drug as well. The antioxidant action of this drug was studied under different oxidant conditions in vitro, and thiobarbituric acid-reactive substances were measured to follow lipid peroxidation. Mexiletine inhibited iron-ascorbate-H2O2-induced lipid peroxidation in brain membranes, liver microsomes and phospholipid liposomes, being most effective in brain membranes. The inhibition was dose- and time-dependent. Mexiletine also inhibited copper-ascorbate-H2O2-induced lipid peroxidation but to a lesser extent. It is concluded that mexiletine has a dual effect toward oxidative injury in brain, both by inhibiting Na+-Ca2+ exchanger-dependent Ca2+ influx and by acting as an inhibitor of lipid peroxidation. However, as this drug is effective at millimolar concentrations, it should be considered less active than natural antioxidants that are effective at micromolar concentrations.

Attenuation of ischemia--reperfusion injury by enalapril maleat.

1. The aim of this study was to investigate the effects of enalapril maleate on ischemia-reperfusion injury of the myocardium, after cardioplegic arrest in isolated guinea pig hearts, in a modified Langendorff model. 2. Animals were subjected to 90 min of normothermic global ischemia, followed by 30 min of reperfusion. Cardioplegic arrest was achieved by administering St. Thomas Hospital cardioplegic solution (STHCS). 3. The hearts were randomly allocated into four groups (n=8 in each group). The first group was utilized as control. In the second group, oral pretreatment was made (0.2 mg/kg enalapril maleat was given twice a day for 10 days). In the third group, enalapril maleat (1 micromol/l) was added to STHCS. In the fourth group, hearts were arrested with enalapril maleat-enriched STHCS, and enalapril maleat-enriched (1 micromol/l) Krebs-Henseleit solution was applied during the reperfusion period. 4. Although the study groups showed better recovery of contractility than did the control group, in the last group, the hearts had the best recovery of left ventricular systolic function, where dp/dt maximum was 89.7+/-6.9% of the preischemic values. Group 1, group 2 and group 3 achieved 44.2+/-4.5%, 79.4+/-5.8% and 68.1+/-6.7% of their preischemic dp/dt values. A similar observation was found for left ventricular developed pressure (LVDP); LVDP values were 52.4+/-2.1% (in group 1), 79.6+/-2.8% (in group 2), 72.8+/-4.6% (in group 3) and 86.7+/-5.8% (in group 4) of control after reperfusion. Creatine kinase leakage was significantly lower and postischemic coronary flows were significantly higher in group 4. 5. We concluded that usage of enalapril maleat in the reperfusion period was more effective for improving myocardial recovery after cardioplegic arrest. The additional protective effects of enalapril maleat not only were by angiotensin-converting-enzyme-inhibition-dependent coronary vasodilation and thiol-dependent limitation of oxidative injury, but could also be related to an oxygen-free-radical-scavenging effect.

Experimental study of the effect of adrenaline tolerance on intestinal ischaemia-reperfusion.

BACKGROUND: Adrenaline tolerance improves survival in animal models of shock. The purpose of this study was to determine the effects of adrenaline tolerance on intestinal ischaemia-reperfusion in a mouse model. METHODS: Adrenaline tolerance was developed by injecting intravenous adrenaline, gradually increasing the dose to 2 mg/kg over 5 days. In experimental animals the superior mesenteric artery was clamped for 120 min and then released. Evans blue dye was given intravenously to all animals to quantify pulmonary microvascular injury. Some 60 min after clamp release, the animals were killed and the effects of reperfusion assessed on tissue samples. RESULTS: Evans blue dye concentrations were significantly higher in animals with intestinal ischaemia-reperfusion than in those having sham intestinal ischaemia-reperfusion or in adrenaline-tolerant mice having intestinal ischaemia-reperfusion or sham ischaemia-reperfusion (P< 0.01). Malonyldialdehyde levels increased significantly in the lung in the intestinal ischaemia-reperfusion group compared with those in the sham ischaemia-reperfusion group (P< 0.001 for liver, lung and small intestine), whereas there was no difference in adrenaline-tolerant animals. There was no significant change induced by adrenaline tolerance in myeloperoxidase levels in any organ. CONCLUSION: Adrenaline tolerance reduced the lung permeability caused by intestinal ischaemia-reperfusion. Catecholamines may play a role in free radical generation induced by ischaemia-reperfusion.

Effects of preoperative fractionated irradiation on left colonic anastomoses in the rat.

PURPOSE: Radiotherapy is frequently used as a (neo)adjuvant to surgery in colorectal cancer patients, and because such therapy could influence the integrity of the anastomosis, we decided to investigate the effect of preoperative irradiation on colonic anastomosis. METHODS: Seventy-two male Wistar rats, weighing 200 to 348 g, were divided into three groups: a control group (I) underwent left colon resection and primary anastomosis (n = 20); a sham irradiated group (II, n = 20); a study group (III) that received fractionated irradiation to the whole pelvis (anterior-posterior pelvic field), for a total dose of 22 Gy, 5.5 Gy per fraction, on four consecutive days with linear accelerator (n = 32). Four days after irradiation, both Groups II and III underwent the same operation as performed in Group I. Within each group, one-half of the animals were anesthetized on the third postoperative day and one-half on the seventh postoperative day. Abdominal wound-healing, anastomotic complications, and anastomotic bursting pressure measurements were recorded. Following these measurements, the anastomotic segment was resected for hydroxyproline content and myeloperoxidase activity. RESULTS: Irradiated animals had more pronounced weight loss during therapy. There were no differences with abdominal wound-healing, intraperitoneal adhesions, and anastomotic complications between groups. At days 3 and 7, mean bursting pressures of the anastomosis were determined at 36.5 and 208 mmHg in Group I, 34.5 and 228 mmHg in Group II, and 25 and 150 mmHg in Group III, respectively (P < 0.01 Group III vs. both Groups I and II on days 3 and 7). The burst occurred at the anastomosis in all animals tested on the third postoperative day and one in Group I (10 percent), none in Group II, and six in Group III (37.5 percent) on the seventh postoperative day. In addition, hydroxyproline content and myeloperoxidase activity was significantly lower in Group III. CONCLUSION: Although preoperative fractionated irradiation significantly decreased the anastomotic bursting pressure and more burst occurred in the anastomotic line on postoperative day 7, the clinical outcome was similar among the groups.

Experimental study of the effect of preoperative 5-fluorouracil on the integrity of colonic anastomoses.

BACKGROUND: Despite curative resection for colorectal cancer, many patients develop recurrence at the primary site or distant organs. These patients are candidates for (neo)-adjuvant chemotherapy. Very little is known about the effect of preoperative 5-fluorouracil (FU) on the healing of colonic anastomoses. The aim of this study was to assess this in a rat model. METHODS: Eighty male Wistar rats, weighing 160-215 g, were divided into three groups; (1) a control group underwent left colon resection and primary anastomosis (n = 20); (2) a sham group received 1 ml saline intraperitoneally (n = 30); (3) a study group received 5-FU intraperitoneally (20 mg kg-1). Both saline and 5-FU injections were given intraperitoneally for 5 days before operation. RESULTS: There was no difference in the rate of wound complications, intraperitoneal adhesions and anastomotic complications among the groups. Three and seven days after operation, mean bursting pressure of the anastomosis was 36.5 and 198 mmHg in group 1, 34 and 200 mmHg in group 2, and 39 and 190 mmHg in group 3 respectively (P not significant). Although the myeloperoxidase and hydroxyproline content were significantly lower after 5-FU therapy (P < 0.01, compared with others), the clinical outcome was similar. CONCLUSION: Preoperative 5-FU consecutive days before operation had no effect on the healing of colonic anastomoses.

Effects of retinoic acid (all-trans and 9-cis) on tumor progression in small-cell lung carcinoma.

The two stereoisomers of retinoic acid (RA), all-trans and 9-cis-RA, are regulators of cell proliferation, differentiation and apoptosis. In this study, the aim was to evaluate the effects of all-trans-and 9-cis-RA on cell growth, proliferation, and on the induction of apoptosis in the human small cell lung carcinoma (SCLC) cell lines NCI-H82 and NCI-H209. The application of various concentrations of all-trans and 9-cis-RA were able to inhibit cell growth and proliferation. Moreover, 3H-thymidine incorporation was inhibited and the number of viable cells decreased, suggesting that all-trans-RA and 9-cis-RA can inhibit cell proliferation in a dose dependent manner. Morphological examinations (light, electron and fluorescence microscopy) demonstrated that both retinoids had profound effects on the induction of apoptosis. Our investigation also showed that, compared to all-trans-RA, 9-cis-RA is a stronger inducer for the inhibition of cell growth and proliferation and that it is more effective in the induction of apoptosis in small cell lung carcinoma cells in culture.

Stable luciferase transfected cells for studying steroid receptor biological activity.

In the course of steroid hormone research, firefly luciferase was used as a reporter gene to construct chimeric cellular models in which the firefly luciferase expression mimics natural hormonal response. Cells containing the endogenous receptor of interest were stably transfected with a reporter gene whose expression is controlled by this endogenous receptor. Based on the detection of luciferase activity in intact cells using a photon-counting camera, various stable transfected cell lines were established. We present potential experimental uses of these cellular models such as for screening new (anti)hormonal molecules. We also show that the hormonal responses can be modulated at any step, suggesting that these stable cell lines may be helpful in studying hormonal interactions. For example, we have detected the antiestrogen activity of molecules able to mediate their effect via a pathway other than the estrogen receptor. Lastly, we show that the detection of luciferase activity in intact living cells is particularly helpful in investigating the variation of the hormonal responses with time. Since chimeric response faithfully reflects hormone (or effector) actions in the cell, we conclude that stable transfected cells can be used in both pharmacological and fundamental studies to investigate different aspects of the endocrine research.

Antiestrogenic effects of all-trans-retinoic acid and 1,25-dihydroxyvitamin D3 in breast cancer cells occur at the estrogen response element level but through different molecular mechanisms.

Most breast tumors show estrogen-dependent growth and are thus susceptible to antiestrogenic therapy. MCF-7 cells, obtained from a human estrogen-dependent breast carcinoma, are widely used for studying the modulation of estrogenic responses by different effectors. All-trans-retinoic acid (RA) and 1,25-dihydroxyvitamin D3 (Vit D3) inhibited estrogen-induced growth of MCF-7 cells and their effect was potentiated by the classical antiestrogen, hydroxytamoxifen. In MCF-7 cells, we found that RA and Vit D3 also inhibited estrogen-induced transcription; this was shown both for an endogenous gene (pS2) and for various exogenous transfected genes. Their inhibitory effect could not be reversed by increasing estradiol concentrations, showing that contrary to classical antiestrogens, they did not compete with estradiol to bind the estrogen receptor (ER). Analysis of the inhibitory mechanisms indicates that RA and Vit D3 receptors can directly or indirectly impair the binding of ER to the estrogen responsive element. The antagonist effect of RA would be found especially at DNA level since it seems to essentially involve an estrogen responsive element. The antagonist effect of Vit D3 would be found especially at the ER level since it seems to concern estrogen binding and dimerization domains of ER. We conclude that the antiestrogenic effects of RA and Vit D3 are similar since they can, via their receptors, interfere with estrogenic action at the estrogen responsive element level but that they are not identical since different molecular mechanisms are involved.

MVLN cells: a bioluminescent MCE-7-derived cell line to study the modulation of estrogenic activity.

We previously established a stable expression system in MCF-7 cells for the detection of (anti)estrogenic activity by assaying the reporter enzyme activity of firefly luciferase. In this cell line (called MVLN), the bioluminescent response can be measured either in the cellular homogenate, or in intact living cells. Here we present various potential experimental uses of this cellular model. First, we used this cell line to screen natural or synthetic molecules classified as full or partial (anti)estrogens and observed that their behavior towards our model was identical to that expected. Moreover, the bioluminescent response was in agreement with the natural responses like cellular proliferation or stimulation of the progesterone receptor. We then demonstrated the inhibitory effects of retinoic acid and 1,25 dihydroxyvitamin D3, two molecules which do not compete with estradiol for its receptor. We thus deduced that with this cell line an "antiestrogenic" effect which occurred at any step of the estrogenic action, might be detected. Finally, we showed that detection of luciferase activity in intact living cells was particularly helpful for investigating the evolution of estrogenic activity. For instance, we observed that long-term treatment of MVLN cells with an antiestrogen irreversibly decreased the bioluminescent response by more than 90%. This phenomenon affected all cells equally and could not be reversed, even by long-term estradiol treatment. We therefore conclude that this chimeric response faithfully reflects estrogenic action in the cell and can be used to develop different aspects of the endocrine research.

Characterization of binding sites for oxyntomodulin on a somatostatin-secreting cell line (RIN T3).

Oxyntomodulin (OXM), a glucagon-containing peptide extended at its C-terminal end by an octapeptide, is a potent inhibitor of gastric acid secretion in rat and man. OXM appears to act on gastric mucosa at least partially through a stimulation of gastric somatostatin release. We have investigated the effects of OXM on a somatostatin-secreting cell line (RIN T3) derived from a radiation-induced rat insulinoma and characterized specific binding sites for this peptide. OXM increased somatostatin release with an ED50 of 2.3 nM. OXM also stimulated the cAMP accumulation in intact RIN T3 cells and adenylate cyclase activity in RIN T3 cell membranes with ED50 values of 0.5 and 11 nM, respectively. On these parameters, glucagon was 10-30 times less potent than OXM. Forskolin, isobutylmethylxanthine, and 8-bromo-cAMP mimicked the effect of OXM on somatostatin release. Specific binding for mono-[125I]OXM was dependent upon time and membrane concentration. Binding of mono-[125I]OXM was inhibited by OXM and glucagon in a concentration-dependent manner, with dissociation constants (Kd) of 4.5 and 43 nM, respectively. The nonhydrolyzable analogs of GTP (guanosine 5',3-O-(thio)triphosphate and guanosine 5' (beta,gamma-imino)triphosphate decreased the binding of mono-[125I]OXM to its binding sites. Covalent cross-linking of mono-[125I]OXM or mono-[125I]glucagon to RIN T3 cell membranes followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated a single radiolabeled band at 63,000 mol wt, which differed from that observed after cross-linking with liver plasma membranes (55,000 mol wt). These results demonstrate the presence of specific high affinity binding sites for OXM in a somatostatin-secreting cell line (RIN T3) and their coupling to adenylate cyclase via guanine nucleotide-binding proteins.

Study of an antiestrogenic effect of retinoic acid in MCF-7 cells.

We recently developed the cellular model MVLN-15 in which estrogenic action can be detected by bioluminescence. Using this cellular model, we characterized the inhibitory effect of retinoic acid on the estrogen-dependent induction of luciferase transcription. We present evidence that i) the inhibitory effect of retinoic acid is not due to a simple competition between retinoic acid and estradiol for binding to the estrogen receptor, ii) a DNA sequence restricted to an estrogen-responsive element (ERE) was sufficient for the antiestrogenic effect of retinoic acid, and iii) retinoic acid does not act via a cryptic AP-1 binding site associated with this ERE. Therefore, we conclude that the antiestrogenic effect of retinoic acid is due to an inhibition of estrogen receptor activity, for example by altering the amount of estrogen receptor protein bound to the ERE or affecting the transcriptional efficiency of this complex.

[Using examples of firefly luciferase gene for the study of biological activities (estrogens, retinoids, phorbol esters)]. / Exemples d'utilisation du gène de la luciférase de Luciole pour l'étude d'activités biologiques diverses (oestrogènes, rétinoïdes, esters de phorbol).

Some possible applications of chimeric cellular models, specifically responding to an effector through firefly luciferase induction are presented with the help of examples in relation with the biological activity of estradiol or retinoic acid, or phorbol ester. A comparison of experiments on either chimeric or natural responses shows that: i) the responses of both type of cellular models are effector concentration-dependent; ii) these concentrations are in the same order of magnitude; partial agonist compounds and antagonist compounds; iv) potencies (EC50) of test-compounds are similarly classified. Moreover we show that a chimeric cellular model allows the observation of interactions between hormone or effector pathways: it allows readily performed kinetic studies and long-term experiments in intact cells that permit to investigate the effect of a given effector, its reversibility and time-dependent action. Therefore, various steps of a cellular signalling pathway involved in the action of an effector may be observed with such a valuable tool.