Vasopressin receptor capacity of human blood peripheral mononuclear cells is sex dependent

Vasopressin receptors were demonstrated on human peripheral blood mononuclear cells (PBMC) by using the radioiodinated analog of d(CH2)5[Tyr(Me2)Thr4Tyr-NH2(9)]OVT (OTA). Binding of this ligand was time-dependent, specific, and saturable. Scatchard analysis of [125I]-OTA binding at equilibrium revealed a dissociation constant of 0.47 +/- 0.17 nM. A considerable sex difference in binding capacity was observed. PBMC from female donors expressed an approximately sevenfold higher receptor density than PBMC from male donors, while no change of Kd was apparent. Throughout the menstrual cycle the maximal binding capacity was relatively constant. Competition studies with vasopressin and oxytocin analogs showed that this putative receptor site on PBMC is comparable in receptor specificity to the human V1 receptor on myometrial tissue and blood platelets, but slightly different from the rat neurohypophyseal hormone receptor classes. Our findings provide further evidence of a remarkable species and sex difference of vasopressin and oxytocin receptors, regarding their ligand selective binding properties. The presence of the putative arginine-vasopressin receptors on PBMC may provide a molecular basis for several arginine-vasopressin induced effects on the chemistry and function of circulating mononuclear cells.     

Endogenous ligands of the benzodiazepine receptor

The benzodiazepine receptor (BZR) is a site on the GABAA-receptor-chloride channel by means of which benzodiazepine and nonbenzodiazepine compounds produce positive or negative allosteric modulation of the channel gating function and which is blocked by BZR antagonists. Whether the BZR is acted upon by one or several endogenous ligands under normal or pathologic conditions is still a controversial issue after 10 years of intensive research. Evidence is provided for the unconventional view that there need not be an endogenous ligand of the BZR; neither to justify the existence of this receptor, nor to explain the effects of exogenous BZR ligands. Out of a number of putative endogenous compounds with affinity for the BZR, the peptide DBI (diazepam binding inhibitor) stands out as a ligand that might act on the BZR in a subset of GABAergic synapses or under particular situations. The low affinity of DBI for the BZR and its distribution in the body suggest, however, that its primary function might not be at the level of the BZR.     

PET imaging of human gliomas with ligands for the peripheral benzodiazepine binding site

Human gliomas were imaged in vivo using ligands for the peripheral-type benzodiazepine binding site (or omega 3 binding site) and positron emission tomography (PET). Although gliomas have a high density of the peripheral-type benzodiazepine binding site, PET scans with a selective ligand for this site, [11C] Ro5-4864, failed to demonstrate higher radioactivity levels in human gliomas than in brain. In vitro studies of surgically removed specimens of human glioma demonstrated little binding of Ro5-4864 but high levels of binding of another selective ligand, PK 11195. Scans with [11C]PK 11195 demonstrated increased radioactivity in glioma compared to brain in 8 of 10 patients. Radioactivity in tumor and the ratios of radioactivity in tumor to that in remote gray and in white matter correlated significantly with the specific activity of [11C]PK 11195, suggesting that accumulation represents saturable high-affinity binding. We conclude that the PK 11195 manifests greater binding than Ro5-4864 to the peripheral-type benzodiazepine binding site on human gliomas and that human gliomas can be successfully imaged using [11C]PK 11195 and PET.     

Effects of central and peripheral type benzodiazepine ligands on thyrotropin and prolactin secretion.

The cold-stimulated thyrotropin (TSH) levels in the rat were decreased by clonazepam (a central type benzodiazepine agonist), diazepam (a mixed agonist), FG 7142 (an inverse central type agonist) and Ro 5-4864 (a peripheral type agonist), clonazepam being the most potent and Ro 5-4864 the least active. Clonazepam and diazepam also decreased while FG 7142 increased prolactin (PRL) levels. Ro 5-4864 did not have any significant action. Clonazepam (1 and 5 mg/kg) and diazepam (15 mg/kg but not 25 mg/kg) decreased even the TRH-induced PRL levels. Only Ro 5-4864 (25 mg/kg) decreased TRH-induced TSH secretion but not significantly. The actions of central type compounds were antagonized by flumazenil but not by PK 11195. The weak effects of Ro 5-4864 were not antagonized by either antagonists. While the peripheral type benzodiazepine agonist only weakly affected the secretion of anterior pituitary hormones, the central type inhibition of TSH appears to be mediated through the hypothalamic TRH and that of PRL rather through the anterior pituitary gland. The sedating (or agitating in case of FG 7142) effect of high doses of benzodiazepine ligands may contribute to the changes in TSH and PRL levels.     

3H-spiroperidol binding to human peripheral mononuclear cells: methodological aspects.

3H-spiroperidol binding to lymphocytes has been proposed as a vulnerability marker for schizophrenia. However, the biological significance and even existence of this "binding site" are still in controversy. Therefore, the present study reevaluated methodological details using a filtration binding assay. The results indicated that some well-known, but obviously uncontrolled pitfalls might contribute to this controversy [e.g., unspecific filter binding, which increased in the presence of (+)-butaclamol, or a variable amount of contaminating granulocytes). Moreover, due to an atypically shaped saturation curve, different mathematical methods to analyze the data were used and compared. The present data should help us to understand the biological relevance of this marker, as viewed in different laboratories.     

体外诱导人外周血单个核细胞向肝样细胞的分化

背景：外周血具有收集方便、供者不需麻醉、无骨髓穿刺痛苦等优点,利用人外周血干细胞移植的方法来获得转分化后的肝样细胞临床应用前景较好。 目的：探讨人外周血单个核细胞在肝细胞生长因子及成纤维细胞生长因子4诱导下分化为肝样细胞的可行性。 设计、时间及地点：细胞学体外对照观察,于2007-05/10在中国科学院大连化物所1806组细胞培养室和大连医科大学附属第一医院中心实验室完成。 材料：外周血由大连市红十字血液中心11名健康志愿献血者提供。肝细胞生长因子、巨噬细胞集落刺激因子、成纤维细胞生长因子4为Peprotech公司产品。 方法：采集志愿者外周血,密度梯度离心法分离外周血单个核细胞,加入RPMI1640液培养2 h后,去除未贴壁细胞。将贴壁细胞分为4组：对照1组加入含140 μmol/L β-巯基乙醇、体积分数为0.1胎牛血清的RPMI1640液培养6 d;对照2组、肝细胞生长因子组、成纤维细胞生长因子组均在其基础上向RPMI1640液中加入5 μg/L巨噬细胞集落刺激因子、0.4 μg/L白细胞介素3。洗涤后,对照组换用含胎牛血清的RPMI1640培养液继续培养20 d,肝细胞生长因子组在其基础上加入20 μg/L肝细胞生长因子,成纤维细胞生长因子组加入10 μg/L成纤维细胞生长因子4。 主要观察指标：诱导分化过程中细胞形态学变化,免疫荧光检测诱导后甲胎蛋白及白蛋白的表达。 结果：刚分离的单个核细胞形态呈较均一的圆形,活细胞率>95%,培养2 h后贴壁细胞呈毛糙的圆形,6 d后肝细胞生长因子组、成纤维细胞生长因子组细胞呈集落样生长,细胞趋向融合,对照组细胞未形成集落,呈单个细胞生长。经诱导培养后,4 d时部分细胞体积变大,向类圆形细胞转变,随培养时间延长,类圆形细胞比例增加,至20 d后细胞数量逐渐减少;对照组培养20 d后多数细胞呈梭形或纤维样,少部分细胞呈圆形。诱导培养后第6,10,14,20天,肝细胞生长因子组、成纤维细胞生长因子组甲胎蛋白、白蛋白均呈阳性表达,对照组各时间点均呈阴性表达。 结论：在肝细胞生长因子及成纤维细胞生长因子4诱导条件下,人外周血单个核细胞具有向肝样细胞分化的潜能。 关键词：外周血单个核细胞;肝细胞生长因子;成纤维细胞生长因子;肝细胞;分化     

Lorazepam and FG 7142 induce tolerance to the DMCM antagonistic effect of benzodiazepine receptor ligands

Mice were given chronic treatment with lorazepam 10 mg/kg PO or FG 7142 40 mg/kg IP once a day for 14 days. The pretreatments with lorazepam and FG 7142 did not change the sensitivity of the mice to the convulsant effect of DMCM. Lorazepam pretreated mice showed a significantly lower sensitivity to the anticonvulsant effects of the benzodiazepine (BZ) receptor ligands lorazepam, ZK 93423, ZK 91296, Ro 15-1788 and ZK 93426 administered acutely by the IP route when challenged with DMCM 24 hr after the last dose of lorazepam. FG 7142 pretreated mice showed a significantly lower sensitivity to the anticonvulsant effect of the two agonists lorazepam and ZK 93423 and to the antagonist Ro 15-1788, whereas the effects of ZK 91296 and ZK 93426 were left unchanged. The reduced DMCM antagonistic effects of the BZ receptor ligands may indicate that these ligands may either have lost some of their affinity to those BZ receptors being responsible for the DMCM-induced seizures or they may have lost some efficacy in allosterically inhibiting DMCM binding or as a third possibility may have lost efficacy at a BZ receptor site downstream to the seizure-inducing center in the brain.     

Age-related changes in peripheral benzodiazepine receptors of human platelets.

Using 3H-PK 11195 as radioligand, the number and affinity of peripheral benzodiazepine receptors in platelets of 15 elderly healthy subjects were compared to those of 15 young subjects. The results showed that the dissociation constant (Kd) was significantly higher in the elderly than in the young subjects, while the density of binding sites did not differ. These findings suggest that the age-related changes in peripheral benzodiazepine receptors may be coupled with secondary changes in their hypothesized functions.     

An analysis of peripheral type benzodiazepine receptors on blood mononuclear cells during high dose steroid treatment of multiple sclerosis

We report here a study of peripheral type benzodiazepine receptors (pBZr) in mononuclear cells (MNC) from blood of patients with multiple sclerosis (MS) during periods of stable and active disease and from normal controls. Most active MS patients were retested in a longitudinal study, both during a treatment with high dose steroids and while medication free. Active MS produces a significant decrease of receptor density compared with the controls whereas remission of the disease shows no effect. Four weeks of steroid treatment restore binding density to normal levels, and two weeks of drug withdrawal result in a small, but significant increase in number of the binding sites compared with the control value. We suggest that the number of pBZr in blood MNC might change during the clinical course and steroid therapy of MS. Paper presented at the National Congress at Sorrento in 1991 and selected by the Editorial Board of the Journal     

Pharmacological modulation of neuropeptides in peripheral mononuclear cells.

The concentrations of beta-endorphin and cholecystokinin were measured in fresh resting peripheral mononuclear cells obtained from rats and human subjects in basal conditions and after different pharmacological treatments. Both in the human and the rat, beta-endorphin concentrations in mononuclear cells, increased after treatment with serotoninergic agonists, decreased after dopaminergic or GABAergic drugs, while the respective antagonists exerted the opposite effect. In vitro, serotoninergic and GABAergic compounds confirmed their roles in the modulation of beta-endorphin in mononuclear cells. Cholecystokinin was never affected by the pharmacological treatments.     

Differential effects of beta-endorphin on cAMP levels in human peripheral blood mononuclear cells

In the present paper we demonstrate that one of the early effects of the opioid peptide beta-endorphin on human peripheral blood mononuclear cells is the induction of a change in the intracellular cAMP level. However, the effect of beta-endorphin on cAMP levels is not uniform; increases as well as decreases in cAMP level are observed. It appears that beta-endorphin is a true modulator of intracellular cAMP level: the peptide will increase cAMP levels in cells with a low baseline level. In contrast, beta-endorphin tends to decrease cAMP levels is cells with a high cAMP concentration. Moreover, beta-endorphin modulates the rise in cAMP induced by beta-adrenergic activation. The effect of beta-endorphin on cAMP level correlates negatively with the magnitude of the change in cAMP level induced by beta-adrenergic activation.     

The contribution of endogenous benzodiazepine receptor ligands to the pathogenesis of hepatic encephalopathy.

The involvement of the gamma-aminobutyric acid A(GABAA) receptor complex in the pathogenesis of hepatic encephalopathy (HE) was examined in galactosamine-treated rabbits with HE caused by fulminant hepatic failure. Radioligand binding to the constituent components of the GABAA receptor complex was unchanged in rabbits with HE. However, partially purified extracts from encephalopathic rabbit brain were approximately three times more potent in inhibiting [3H]Ro 15-1788 binding to benzodiazepine (BZ) receptors than extracts from control rabbits. The inhibition of radioligand binding to the BZ receptor produced by these extracts was competitive and reversible and was significantly enhanced by GABA. Further purification of these extracts by high-performance liquid chromatography (HPLC) indicated that the inhibitory activity was localized in several peaks, some of which had retention times corresponding to 1,4-benzodiazepine standards. The presence of diazepam in these extracts was confirmed using mass spectroscopy. Both mass spectroscopic and radiometric techniques demonstrated that the concentration of diazepam in brain extracts from encephalopathic rabbits was approximately 4 times greater than control extracts. These findings link the presence of BZ receptor agonists to the development of a neuropathological condition, thereby providing a rational basis for the use of BZ receptor antagonists in the management of HE in man.     

Expression of nerve growth factor receptors by human peripheral blood mononuclear cells

In the rat, nerve growth factor (NGF) has been shown to affect immune reactivity by binding to cell surface receptors on a subpopulation of splenic mononuclear cells. This binding occurs in a specific and saturable fashion to what appear to be low-affinity (type II) NGF receptors (NGFR). Immunofluorescence studies here showed that NGFR are also present on a proportion of human peripheral blood mononuclear cells (PBMC). Equilibrium binding studies demonstrated that the binding of NGF to its receptors on PBMC occurs with a single equilibrium binding constant (mean) of 2.11 X 10(-9) M. The number of receptors per cell was determined to be approximately 6.94 X 10(3) receptors/cell. These results would suggest a role for NGF in the regulation of immune function in man, as well as in animals.     

Bidirectional effects of benzodiazepine receptor ligands against picrotoxin- and pentylenetetrazol-induced seizures

Summary The dose response curves of picrotoxin-induced seizures and pentylenetetrazol-induced seizures were shifted to the right by the benzodiazepine (BZ) receptor agonist lorazepam, and to the left by the inverse agonists, DMCM, ZK 90886, FG 7142 and CGS 8216. The BZ receptor antagonists ZK 93426 and Ro 15-1788 had no effect on the dose response curves. The anticonvulsive action of lorazepam and the proconvulsive action of DMCM against picrotoxin-induced seizures and against pentylenetetrazol-induced seizures was inhibited by low doses of ZK 93426 and Ro 15-1788.These results indicate that the bidirectional effects of benzodiazepine receptor ligands on picrotoxin and pentylenetetrazol induced seizures is actually mediated through benzodiazepine receptors.     

Altered cannabinoid receptor mRNA expression in peripheral blood mononuclear cells from marijuana smokers

We studied, using RT-PCR, the relative expression of cannabinoid receptor (CBR) mRNA in peripheral blood mononuclear cells (PBMC) from different donor groups. Cells from normal donors expressed a CB2 mRNA level threefold higher than CB1 across all age, gender or ethnicity groups, and amplicons were of the same size in all donors. However, cells from marijuana users expressed higher levels of CBR mRNA, but with a preserved CB1/CB2 ratio of 1:3. CBR gene products were also studied following short-term mitogen activation in vitro. CB1 expression decreased following mitogen stimulation when compared to the time-matched medium only cells while the expression of CB2 mRNA remained unchanged. These studies suggest that marijuana smoking and immune activation can alter the basal levels of CB1 and CB2 in PBMCs.     

Bidirectional modulation of stimulated cortical acetylcholine release by benzodiazepine receptor ligands

In vivo microdialysis was used to determine the ability of benzodiazepine receptor (BZR) ligands to modulate stimulated cortical acetylcholine (ACh) efflux in awake, freely-moving Fischer-344/BNNia rats. Cortical ACh efflux was reliably enhanced during presentation of a complex stimulus (exposure to darkness coupled with presentation of a small amount of palatable food) in animals entrained with that stimulus. Administration of the BZR selective inverse agonist ZK 93 426 (5.0 mg/kg, i.p.) potentiated the ability of the darkness/food stimulus to enhance efflux, whereas administration of the BZR full agonist, chlordiazepoxide (5.0 mg/kg, i.p.) blocked the enhancement. The interaction of the BZR ligands with the entrained stimulus in affecting cortical ACh efflux was not secondary to effects on motor activity. These results, combined with results from a previous study, suggest that modulation of cortical ACh efflux by BZR ligands is bidirectional and dependent on the level of activity within cortical cholinergic neurons. This relationship enables the trans-synaptic stimulation of cortical ACh transmission by BZR inverse agonists to be most effective during behavioral activities which recruit the basal forebrain cholinergic system.     

Translocator protein (18 kDa)/peripheral benzodiazepine receptor specific ligands induce microglia functions consistent with an activated state

In the brain, translocator protein (18 kDa) (TSPO), previously called peripheral benzodiazepine receptor (PBR), is a glial protein that has been extensively used as a biomarker of brain injury and inflammation. However, the functional role of TSPO in glial cells is not well characterized. In this study, we show that the TSPO-specific ligands R-PK11195 (PK) and Ro5-4864 (Ro) increased microglia proliferation and phagocytosis with no effect on migration. Both ligands increased reactive oxygen species (ROS) production, and this effect may be mediated by NADPH-oxidase. PK and Ro also produced a small but detectable increase in IL-1β release. We also examined the effect of PK and Ro on the expression of proinflammatory genes and cytokine release in lipopolysaccharide (LPS) and adenosine triphosphate (ATP) activated microglia. PK or Ro had no effect on LPS-induced increase of pro-inflammatory genes, but they both decreased the ATP-induced increase of COX-2 gene expression. Ro, but not PK, enhanced the LPS-induced release of IL-1β. However, Ro decreased the ATP-induced release of IL-1β and TNF-α, and PK decreased the ATP-induced release of TNF-α. Exposure to Ro in the presence of LPS increased the number of apoptotic microglia, an effect that could be blocked by PK. These findings show that TSPO ligands modulate cellular functions consistent with microglia activation. Further, when microglia are activated, these ligands may have therapeutic potential by reducing the expression of pro-inflammatory genes and cytokine release. Finally, Ro-like ligands may be involved in the elimination of activated microglia via apoptosis.     

Sigma receptor ligands protect human retinal cells against oxidative stress

This study was undertaken to investigate the role of sigma receptors during the oxidative damage on human retinal pigment epithelial cells, and to assess whether sigma receptor ligands enhance survival and protect DNA of cells challenged by oxidative stress. Pretreatment with PRE-084, a sigma1 receptor agonist, resulted in significant increased viability in a dose-related manner. DNA damage induced by oxidative insult was significantly lower with PRE-084. The effects of PRE-084 were antagonized by pretreatment with sigma1 receptor antagonists (NE-100 and BD1047), but interestingly were synergized by cotreatment with BD1047 that also presented an affinity for the sigma2 receptor. The results suggest that sigma1 receptors play an important role against retinal damage, even though sigma2 receptor involvement cannot be excluded.