Specific binding of [3H]heparin to bovine granulosa cell membranes

Bovine granulosa cell membranes from small (SFM) and large (LFM) antral follicles were incubated with [3H]heparin, a commercial radioactively labeled glycosaminoglycan (GAG). Binding was specific, reversible, saturable, and dependent on time, pH, ionic strength and divalent cations. SFM exhibited different [3H]heparin binding characteristics compared to LFM. The addition of a physiological concentration of calcium (2 mM) yielded significant differences (P less than 0.02) in [3H]heparin binding for SFM (87 590 +/- 4206 dpm/10(6) cells) compared to LFM (55 230 +/- 2816 dpm/10(6) cells). SFM and LFM showed maximum [3H]heparin binding at pH 6.5 and pH 5.5, respectively. Increasing the ionic strength by addition of 0.07-2.0 M NaCl interfered with binding. Addition of unlabeled heparin (0.1-100 micrograms/ml) displaced [3H]heparin bound to SFM and LFM in a dose-dependent manner, as did dextran sulfate, a non-GAG sulfated branched polysaccharide. Commercial chondroitin sulfate ABC displaced the bound [3H]heparin only at doses between 50 and 500 mg/ml. GAGs purified from FF suppressed binding 39% at a concentration of 5.9 mg/ml. Photomicrographs of fluorescein-labeled heparin bound to granulosa cells showed localized areas of heparin binding to the cell surface. These experiments demonstrated that the GAG heparin specifically bound to bovine granulosa cell membranes, and that significant differences existed between the binding characteristics of SFM and LFM.     

Stimulation of specific binding of [3H]-progesterone to bovine luteal cell-surface membranes: specificity of digitonin.

Non-genomic actions of progesterone have been described in the ovary, and luteal membranes of several species have been shown to possess specific binding sites for [3H]-progesterone. However, binding of radiolabelled progesterone to luteal membranes was demonstrable only in the presence of digitonin. Digitonin is a non-ionic detergent which is thought to act by forming one-to-one complexes with certain sterols. It is also a cardiotonic agent, inhibiting (Na+-K+) ATPase activity by interaction with the extracellular (ouabain/K+) binding site. We therefore investigated which properties of digitonin were responsible for its stimulatory actions on progesterone binding to bovine luteal membranes. A range of compounds with detergent, cardiotonic and or cholesterol-complexing activities were tested for their effects on [3H]-progesterone binding to bovine luteal membrane fractions, and on haemolysis of rat erythrocytes. Stimulation of progesterone binding to luteal membranes was highly specific for digitonin, and a number of ionic and non-ionic detergents, cardenolides, saponins and cholesterol-complexing reagents tested failed either to stimulate [3H]-progesterone binding to bovine luteal membranes in the absence of digitonin, or to inhibit binding specifically in the presence of digitonin. When digitonin was first reacted with excess cholesterol or pregnenolone to form the respective digitonides, stimulatory activity was greatly reduced, suggesting that the ability of digitonin to interact with (an) endogenous steroid(s) may be important in its action. High performance liquid chromatography (HPLC)-mass spectrometry of commercially available digitonin preparations indicated the presence of numerous minor impurities in most commercial digitonin preparations. Three major UV-absorbing peaks were isolated and characterised by mass spectrometry: all stimulated progesterone binding to bovine luteal membrane receptors in a dose-dependent manner, though to differing extents. Our data suggest that the unique action of digitonin on luteal membrane progesterone receptors is not related to its detergent or cardiotonic properties, but appears to be related to its ability to complex with membrane sterols.     

Comparison of the binding of human chorionic gonadotropin to isolated bovine luteal cells and bovine luteal plasma membranes.

The specific binding of [125-I]iodohCG to intact luteal cells obtained from bovine corpus luteum by enzymatic treatment, or to purified plasma membranes obtained from bovine corpus luteum has been compared. It was a saturable process with respect to the [125-]iodohCG concentration. Specific binding could be detected at concentrations as low as 1.8 ng of HCG per ml and saturation achieved at 92 ng/ml. The [125-I]iodohCG specifically bound to the luteal cells or to the plasma membranes was displaced by increasing concentrations of native hCG. Subunits hCGalpha and beta had respectively 200- and 800-fold less activity than hCG. Four to 10 times more ovine LH than hCG was required to displace an identical amount of bound [125-I]iodohCG. The binding of hCG to its receptor site was a function of time and temperature. The affinity of hCG for its receptor sites in luteal cells or plasma membranes of luteal cells was similar (dissociation constants of 5.3 and 3.8 times 10- minus 10 M, respectively). The number of sites per luteal cell was 5 times 10-4 and the capacity of plasma membranes to bind hCG was 140 fmol per mg of protein at saturation. The data does not, however, allow a comparison between the number of binding sites in the two preparations. It is concluded that the enzymatic treatment necessary to obtain a suspension of viable luteal cells does not affect the kinetic characteristics of the binding of hCG to receptor sites since they are similar to those of plasma membranes not treated with proteolytic enzymes.     

Characteristics of somatotropic growth hormone binding to homologous liv plasma membranes

The binding of 125I-labeled oGH, rGH and hGH to homologous liver membranes is compared to define the characteristics of somatotropic hormone-receptor interactions. The effect of pH and cations and the time-course of binding of these different GHs was similar when binding was determined in liver membranes from the homologous species. The time-course of binding was characterized by stable binding by 90-120 min of incubation at 22 degrees C with relatively high nonspecific binding that continued to increase with time. This resulted in a peak of specific binding for each hormone. Addition of excess unlabeled hormone at 30 min of the binding incubation resulted in partial displacement of the bound GH. The binding of rGH was somewhat dependent upon the presence of divalent cations (Mg2+, Ca2+) in the incubation medium. In the absence of added divalent cation, a small but appreciable amount of binding was detected at pH 7-8. With 10 mM calcium, binding increased several-fold with a definite peak of rGH binding at pH 4-5. Both oGH and hGH exhibited a broad peak of binding at pH 5-7. Monovalent cations (Na+, K+) were either ineffective in enhancing GH binding or produced a slight enhancement at higher concentrations (100 mM). Competitive displacement curves confirm the somatotropic nature of the GH-receptor interaction. Human P1 was ineffective in competing for GH binding. We conclude that 'somatotropic' growth hormone binding to its homologous liver receptor exhibits phylogenetically consistent and distinguishing characteristics of binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solubilization and characterization of high-affinity [3H]serotonin binding sites from bovine cortical membranes.

High-affinity [3H]serotonin binding activity has been solubilized from bovine cerebral cortical membranes by using Triton X-100, Tween-80, and octyl-beta-D-glucopyranoside. This mixture of detergents solubilizes the high-affinity [3H]serotonin binding activity present in crude membrane preparations with retention of 75-90% specific binding. The detergent mixture was chosen because it can easily be removed from the solubilized fraction by dialysis and polystyrene bead adsorption, thus permitting further purification and isolation of the binding sites. Saturation analysis reveals multiple components of high-affinity [3H]serotonin binding. In crude bovine cortical membranes, at least two binding components are present. A higher-affinity binding component, as defined from curvilinear Scatchard plots, has a Kd for [3H]serotonin of 1-3 nM, whereas a lower-affinity component has a Kd of 10-20 nM. In the solubilized preparation, only a single class of binding sites is apparent, with a Kd of 50-100 nM. Removal of detergents by dialysis and polystyrene bead adsorption results in restoration of the curvilinear Scatchard plot with apparent Kds similar to those observed in crude membrane preparations and with increased Bmax values for each component. [3H]Serotonin binding activity in the solubilized preparation is stable to Sephacryl S-300 column chromatography and to glycerol gradient sedimentation. Saturation analysis of the peak binding fractions from both these procedures once again yields curvilinear Scatchard plots, indicating that the multiple high-affinity binding components are preserved and migrate together. The molecular weight, Stokes radius, and frictional coefficient of the binding site(s) have been calculated. After detergent removal the solubilized material shows many of the characteristics usually attributed to S1 receptors, such as high affinity for [3H]serotonin and its analogs and low affinity for serotonin antagonists.     

Analysis of [3H]estradiol binding to nuclei prepared from epididymides of sexually immature intact rabbits

The estrogen receptor that is present in the epididymis of sexually immature rabbits is capable of entering nuclei in the time- and temperature-dependent manner characteristic of such reactions. Nuclear translocation reaches a maximum after 1 h of incubation at 23°C. Even short incubations (15 min) at 37°C destroy the cytoplasmic receptor and as a consequence nuclear translocation at this temperature is precluded. We have noted that a small, but demonstrable, amount of receptor enters nuclei at 0°C. Nuclear translocation is absolutely dependent upon estradiol binding to the receptor as shown by the fact that inhibitors of estradiol binding to the receptor (unlabeled estradiol, estrone and estriol) prevent translocation. Compounds that do not prevent estradiol binding to the receptor (5α-dihydrotestosterone, progesterone and cortisol) have no effect on the translocation of the estradiol-receptor complex to nuclei. Nuclei incubated with [³H]estradiol in the absence of receptor did not take up the hormone. Nuclear binding of estradiol falls into two categories: (a) that which is readily extractable with 0.4 M KCl; and (b) that which is resistant to KCl extraction. The resistant fraction is further subdivided into a fraction from which label can be readily extracted with ethanol and an ethanol-resistant residual fraction. The KCl-soluble fraction represents about 30% of the total radioactivity associated with nuclei. Acceptor sites for the estradiol-receptor complex are not limited to target cell nuclei since the complex is apparently bound to nuclear constituents in two presumptive nontarget tissues (spleen and skeletal muscle). However, the number of acceptor sites in target cell nuclei is demonstrably greater than in nontarget cell nuclei.     

Involvement of SAPK/JNK in prostaglandin E(1)-induced VEGF synthesis in osteoblast-like cells

We previously reported that prostaglandin E₁ (PGE₁) activates both p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase via cAMP-dependent protein kinase in osteoblast-like MC3T3-E1 cells, and that p38 MAP kinase but not p42/p44 MAP kinase is involved in PGE₁-induced synthesis of vascular endothelial growth factor (VEGF). In the present study, we investigated the involvement of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in the PGE₁-induced VEGF synthesis in MC3T3-E1 cells. PGE₁ induced the phosphorylation of SAPK/JNK. SP600125, a specific inhibitor of SAPK/JNK, markedly reduced the PGE₁-induced VEGF synthesis. Forskolin, a direct activator of adenylyl cyclase, elicited the phosphorylation of SAPK/JNK, and 8bromo-cAMP, a plasma membrane-permeable cAMP analogue-stimulated VEGF synthesis was significantly reduced by SP600125. SP600125 suppressed the PGE₁-induced phosphorylation of SAPK/JNK without affecting the phosphorylation of p38 MAP kinase induced by PGE₁. The phosphorylation of c-Jun induced by PGE₁ was also inhibited by SP600125. SB203580, a p38 MAP kinase inhibitor, failed to reduce the PGE₁ induced phosphorylation of SAPK/JNK. A combination of SP600125 and SB203580 suppressed the PGE₁-stimulated VEGF synthesis in an additive manner. These results strongly suggest that PGE₁ activates SAPK/JNK in osteoblasts, and that SAPK/JNK plays a part in PGE₁-induced VEGF synthesis.     

Quantitative light microscopic autoradiographic study on [3H]leukotriene C4 binding to nonpregnant bovine uterine tissue

Mammalian uteri contain both lipoxygenase and cyclooxygenase pathways of arachidonic acid metabolism. Sulfidopeptidyl leukotrienes formed by the lipoxygenase pathway can stimulate uterine contractions and play a role in uterine preparation for implantation. These actions of leukotrienes are perhaps mediated by binding to specific receptors. To understand the cellular basis of leukotriene C4 action, the present quantitative light microscopic autoradiographic study was undertaken on nonpregnant bovine uterine tissue. The results demonstrated that the circular and elongated myometrial smooth muscle, uterine vascular smooth muscle, stromal cells of endometrium, and fibroblasts of perimetrium, but not the endometrial glands, vascular endothelium, and erythrocytes in lumen of arterioles, contained specific silver grains after incubation with [3H]leukotriene C4. The number of grains per 100-micron2 areas were similar in circular and elongated myometrial smooth muscle (P greater than 0.05), which was higher than in other uterine cells (P less than 0.05-0.01). The grains in all cells were greatly reduced after coincubation with excess unlabeled leukotriene C4, but not with leukotriene A4, leukotriene B4, leukotriene D4, leukotriene E4, prostaglandin E2, prostaglandin F2 alpha, or prostacyclin. In conclusion, leukotriene C4 may regulate both uterine cells and uterine vasculature and exert contractile and noncontractile actions via the specific leukotriene C4-binding sites present in different cell types.     

Age-correlated decline in [3H]tiagabine binding to GAT-1 in human frontal cortex

BACKGROUND AND AIMS: In spite of the fact that GABA is a significant transmitter, little is known about the GABA system in aging, compared with other transmitter systems. [(3)H]tiagabine is a ligand for GABAergic neurons, which binds with 10-fold higher affinity to the GABA uptake site than [(3)H]nipecotic acid. The aim of this study was to study the binding of [(3)H]tiagabine to the GABA transporter 1, GAT-1, in human frontal cortex and cingulate cortex from individuals of varying ages. METHODS: [(3)H]tiagabine binding experiments were conducted on post-mortem brain tissue from 19 individuals (age range 17-78 years) without known neurological or psychiatric disorders. Binding data vs age and postmortem interval was analysed by Pearson correlation. RESULTS: The density of [(3)H]tiagabine binding to GAT- 1 decreased significantly with increasing age in the frontal cortex, whereas binding affinity was unchanged. No significant alterations in binding parameters were observed in the cingulate cortex. No correlation was found between post-mortem delay and the number of [(3)H]tiagabine binding sites. CONCLUSIONS: According to the present study, presynaptical alterations in the GABA system are correlated with aging in the frontal cortex of the human brain. Further studies involving a broader range of brain regions seem warranted, to confirm the present findings and to enlarge knowledge about the GABA system in aging.     

Influence of steroid hormone treatments or pregnancy on [3H]prazosin and [3H]rauwolscine binding to myometrial alpha-adrenoceptors in the ewe.

The adrenergic antagonists [3H]prazosin and [3H] rauwolscine were used to identify alpha 1- and alpha 2-adrenoceptors respectively in the ovine myometrium. Ewes were allocated to four groups according to steroid hormone treatments or physiological status, namely ovariectomized ewes either as untreated controls, treated with oestradiol-17 beta or progestagen plus oestradiol-17 beta, and pregnant ewes at mid-gestation. Binding of both [3H]prazosin and [3H]rauwolscine to membrane preparations from the ovine myometrium was saturable, of high affinity and rapidly reversed by phentolamine (10 mumol/l). Based on the relative order of potency of selected adrenergic agonists and antagonists, the myometrial binding sites labelled by [3H]prazosin and [3H]rauwolscine were characterized as alpha 1- and alpha 2-adrenoceptors respectively. Saturation binding studies with [3H]prazosin showed that the number of alpha 1-adrenoceptors was low (maximal binding capacity, Bmax, between 19 and 24 fmol/mg protein) and there were no noticeable differences between the animal groups. Moreover, the equilibrium dissociation constant (Kd) did not vary significantly between groups (Kd between 0.10 and 0.17 nmol/l). In contrast, saturation binding studies with [3H]rauwolscine revealed the presence of a high number of alpha 2-adrenoceptors. Values of Bmax were far higher in the pregnant ewes (1096 +/- 241 fmol/mg protein; means +/- S.D.) than in any of the non-pregnant ovariectomized ewes. For these latter groups, the highest Bmax values were found in the group treated with both progestagen and oestrogen (382 +/- 77 fmol/mg protein) compared with treatment with oestrogen alone (101 +/- 8 fmol/mg protein) or with controls (82 +/- 12 fmol/mg protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localized binding of [3H]muscimol to synapses in chicken retina

Binding sites for [3H]muscimol, an analogue of gamma-aminobutyric acid (GABA) were localized in the synaptic layers of chicken retina by light microscopic and electron microscopic autoradiography. Light microscopic autoradiography of cryostat sections incubated in [3H]muscimol or [3H]GABA revealed identical binding patterns: a band over the inner plexiform layer (IPL) and a band over the outer plexiform layer (OPL). This binding pattern differed from the uptake pattern for [3H]GABA: labeling over horizontal, amacrine, and ganglion cell bodies as well as very intense labeling over lamina 5 in the proximal IPL. Statistical analysis of electron microscopic autoradiography data from the IPL indicated that only amacrine synapses bind [3H]muscimol (i.e., make GABAergic synapses). Processes of amacrine, bipolar, or ganglion cells can be postsynaptic to these amacrine synapses. The highest concentration of synapses binding [3H]muscimol occurred in laminae 2 and 4 of the IPL and not in lamina 5 as might be expected from the density of [3H]GABA uptake. In the OPL, [3H]muscimol binding occurred over specialized junctions proximal to photoreceptor terminals. In cone receptor terminals, [3H]muscimol binding was suspected near horizontal cell dendrite/receptor terminal membranes lateral to the synaptic ribbon, supporting the hypothesis that horizontal cells are involved in a GABAergic feedback loop with cone terminals. We conclude that the synaptic binding pattern provides a more accurate concept of GABAergic synaptic interaction than does the uptake pattern for [3H]GABA because the two patterns in the IPL are not related.     

Diazepam receptor: specific nuclear binding of [3H]flunitrazepam.

Autoradiographic localization of [3H]flunitrazepam in nuclei of the rat cerebral cortex was further confirmed by biochemical analysis of specific nuclear binding. Highly purified rat cerebral cortex nuclei were shown to bind [3H]flunitrazepam specifically. The Kd(app) for nuclear binding was 28 nM for the nuclei compared with a Kd(app) of 1.1 nM for binding of [3H] flunitrazepam to synaptosomal membrane fractions of the same tissue. Inhibition of the nuclear binding with inosine and hypoxanthine was greater than inhibition of the synaptic membrane fractions. These results lead to to conclude that specific binding may occur at both the synaptic membrane and the nuclear levels and that different endogenous ligands may compete at each site for binding. Furthermore, the possibility exists for translocation and alteration of the bound ligand complex from membrane site to nuclear site.     

Nerve growth factor enhances [3H]nicotine binding to a nicotinic cholinergic receptor on PC 12 cells.

Although nicotinic cholinergic agonists have functional effects on PC 12 cells, radioligand-binding sites have not been detected. We, therefore, studied PC 12 cells incubated in the presence of nerve growth factor (NGF) and determined that specific [3H]nicotine-binding sites were induced approximately 2.5-fold in the presence of NGF (50 or 100 ng/ml). Specific binding was maximal between the first (100 ng/ml NGF) and seventh (50 ng/ml NGF) days of treatment and was stable for 2 weeks with addition of NGF every 3 days. Using intact cells, average association and dissociation rates for [3H]nicotine were 0.00021 min-1 nM-1 (n = 2) and 0.048 min-1 (n = 2), respectively, at 4 C, yielding an average apparent Kd of 229 nM. At 22 C, stable equilibrium was not attained during association studies. A similar Kd value for broken cell preparations was obtained by kinetic analysis (i.e. an average association rate of 0.00042 min-1 nM-1 and dissociation rate of 0.087 min-1), yielding an average Kd value of 207 nM (n = 2) at 4 C. By saturation binding analysis of intact cells, an average Kd of 292 nM (n = 2) and a binding capacity (Bmax) of 15,118 molecules/cell were obtained. [3H]Nicotine binding was inhibited on an equimolar basis by L-(-)nicotine and N-methylcarbamylcholine. D-(+) Nicotine was 7-fold less potent, whereas alpha-bungarotoxin, mecamylamine, and atropine showed no significant inhibition. [3H]Nicotine binding was also inhibited quantitatively by mono-specific polyclonal antibodies raised against the predicted alpha 3-subunit sequence (amino acids 130-139) of the rat neuronal nicotinic cholinergic receptor. This study represents the first biochemical characterization of NGF-stimulated nicotine-binding sites on PC 12 cells and confirms previous evidence of the presence of functional nicotinic cholinergic receptors on these cells.     

High-affinity binding of the antiestrogen [3H]tamoxifen to the 8S estradiol receptor

The interaction of tamoxifen (ICI 46,474), a synthetic antiestrogen, with uterine cytosol proteins of immature calf and rat has been studied directly using the tritiated compound labeled with a high specific activity. The binding complexes were measured by the dextrancoated charcoal, protamine sulfate and hydroxyapatite assays. Scatchard plots revealed a single class of high-affinity (KD congruent to 1.7 nM) binding sites, with a binding capacity similar to that of estradiol. Competitive experiments showed the same binding specificity for estrogens and antiestrogens. Sucrose gradient analysis revealed an 8S binding protein which could be partially proteolysed by trypsin into a 4S binding protein. Kinetic studies showed that the association rate of tamoxifen was 5 times lower than that of estradiol and reacted according to a second order kinetics. The first-order kinetics of dissociation was considerably higher than that of estradiol, giving a half-dissociation time of 20--40 min at 0--2 degrees C. In some cases tamoxifen displayed two slopes of dissociation, but the proportion of the slow-dissociating complex was always inferior to that found with estradiol. In contrast to estradiol, the kinetic constants ratio (k-/k+) gave a calculated dissociation constant, similar to that determined in equilibrium conditions (KD), agreeing with a simple reactional scheme. We conclude that the antiestrogen tamoxifen binds directly to the 8S cytosol receptor for estrogens and not to another receptor for the antagonists. In contrast to estradiol, the antagonist is rapidly dissociated from the receptor sites and is unable to protect them against thermal inactivation. The affinity of tamoxifen for its receptor sites as determined directly is surprisingly high when compared to its affinity evaluated indirectly by competitive experiments. It is then suggested that the two ligands either bind on two different sites of the same protein or induce a different conformational change of the same binding site.     

Alpha-adrenergic receptors in rat myocardium. Identification by binding of [3H]dihydroergocryptine.

[3H]Dihydroergocryptine ([3H]DHE) binds to sites in membranes derived from rat myocardium that have the characteristics expected of alpha-adrenergic receptors. The binding is saturable with 41 fmol [3H]DHE bound per mg of protein and of high affinity with KD = 2.9 nM. The binding is rapid and readily reversible. Adrenergic agonists compete with [3H]DHE for binding in the order: epinephrine greater than norepinephrine greater than isoproterenol; and adrenergic antagonists compete for binding in the order: phentolamine greater than propranolol. For comparison, (-)[3H]dihydroalprenolol [(-)[3h]dha] was used to bind to sites in the same membrane preparations having characteristics of beta-receptors. The number and affinity of beta-receptors were quite similar to those of the alpha-receptors with 46 fmol (-)[EH]DHA per mg protein bound at saturation and KD = 2.5 nM. These techniques allowed identification of both beta- and alpha-adrenergic receptors in membranes derived from isolated atria, right ventricular free walls, and left ventricles including interventricular septa. This is the first report documenting direct identification of myocardial alpha-receptors by radioligand-binding techniques and complements the literature previously reporting myocardial inotopic and electrophysiological responses to alpha-adrenergic stimulation.     

Induction of membrane-type matrix metalloproteinase-1 stimulates angiogenic activities of bovine aortic endothelial cells

Matrix metalloproteinases (MMPs) have been reported to play critical roles in endothelial cell migration and matrix remodeling during the angiogenic process. Among these MMPs, membrane-type MMP-1 (MT1-MMP) is an important molecule that can trigger the invasion of tumor cells by activating MMP-2 on their plasma membrane. However, the precise involvement of MT1-MMP in the angiogenic process has not been determined. To investigate the roles of the MT1-MMP by the matrix remodeling of endothelial cells, MT1-MMP expression vector was transfected into bovine aortic endothelial cells (BAECs). Increased expression of MT1-MMP in BAECs enhanced the activation of MMP-2, invasion and migration of BAECs. Moreover, the capacity of tube formation was increased in MT1-MMP transfectants. However, cotransfection with antisense MT1-MMP expression vector abolished the effects of MT1-MMP overexpression. These observations indicate that MT1-MMP is involved in the angiogenic process of endothelial cells in vitro. This revised version was published online in June 2006 with corrections to the Cover Date.     

Regulation of endothelial progenitor cells by prostaglandin E1 via inhibition of apoptosis

Bone marrow derived endothelial progenitor cells (EPC) improve endothelial function and neoangiogenesis. Prostaglandin E1 (PGE1) is used for the treatment of patients with peripheral artery disease (PAD). However, the molecular effects are only partially understood. Treatment of C57/Bl6 mice with PGE1, 10 microg/kg BW increased the number of circulating Sca-1/VEGFR-2 positive EPC in the blood compared to vehicle (122+/-7% and 119+/-6% after 10 and 20 days). EPC in the bone marrow were upregulated to 125+/-11% (10 days) and 142+/-15% (20 days). PGE1 increased DiLDL/Lectin positive spleen-derived EPC to 170+/-20% and 174+/-14% after 10 and 20 days. Treatment with PGE1 enhanced in-vivo neoangiogenesis by 2-fold (disk assay, 218+/-27%). PGE1 enhanced the SDF-1 induced migratory capacity per number of EPC to 140+/-11%, 146+/-22% and 160+/-16% after 10, 14 and 20 days. Greater migratory capacity was associated with upregulation of expression of telomere repeat-binding factor (TRF2). EPC of PGE1-treated mice were characterized by reduced apoptosis. Similarly, PGE1 prevented H(2)O(2)-induced apoptosis in cultured human EPC. The effect is mediated by PI3-kinase. The effects of PGE1 on EPC were completely prevented by co-treatment with the NO-inhibitor L-NAME, 50 mg kg(-1) p.o. Treatment with the prostaglandin I2 derivative iloprost (10 microg/kg BW, 20 days) did not alter EPC numbers or function. Physical exercise is the basis of the treatment of patients with PAD. Voluntary running increased EPC numbers in mice. Treatment with PGE1 resulted in an additional increase of Sca-1/VEGFR-2- and DiLDL/lectin positive EPC as well as migration. n=10-24 for all groups, all effects p<0.05. In summary, prostaglandin E1 increases the number of EPC in the blood and the bone marrow in mice. The effect is additive to physical exercise, depends on nitric oxide and is characterized by reduction of PI3-kinase mediated apoptosis. PGE1-mediated upregulation of EPC is associated with improved EPC function and enhanced angiogenesis.     

Interleukin-1 in multiple myeloma: producer cells and their role in the control of IL-6 production

We studied the role of interleukin (IL)-1β in patients with multiple myeloma. By in situ hybridization and immunochemistry, myeloid and megakaryocytic cells expressed high levels of the IL-1β gene and produced IL-1β. Myeloma cells less potently expressed the IL-1β gene and IL-1β protein. IL-1β gene expression was not constitutive since it was detected in the bone marrow myeloma cells of two patients, unlike circulating tumoural cells. In addition, nine myeloma cell lines failed to express the IL-1β gene and this expression could not be induced by 12 different cytokines. We demonstrated that IL-1 was mainly responsible for IL-6 production in the tumoural environment through a PGE₂ loop. In fact, an IL-1 receptor antagonist (IL-1RA) blocked PGE₂ synthesis and IL-6 production by 80%; this blockage could be reversed by adding synthetic PGE₂. Similar findings were found with indomethacin, an inhibitor of cyclooxygenase that blocks PGE₂ synthesis. Taken together, these data emphasize the possibility of blocking IL-1 by using IL-1RA or other antagonists in order to block IL-6 production, which is a major tumoural survival and proliferation factor.