Immunohistochemical expression of cyclin T1 in a case of AIDS-related cachexia.

The expression of cyclin T1 in an autoptic case of AIDS-related cachexia was investigated by immunohistochemistry. When contrasted with normal human tissues, a very similar pattern of expression was found. However, a peculiar distribution of cyclin T1 was noticed in the brown fat and in lymph nodes affected by AIDS-associated lymphadenopathy.     

Venous dilator effect of apelin,an endogenous peptide ligand for the orphan APJ receptor,in conscious rats

Apelin is an endogenous depressor peptide for the G protein-coupled APJ receptor. Our hypothesis is that apelin is a venodilator, and it reduces mean circulatory filling pressure (MCFP; index of venous tone). Dose–response curves of apelin (10, 20 and 40 nmol/kg) or vehicle (0.9% NaCl) were constructed in two groups each of conscious, unrestrained rats: unblocked rats and rats pretreated with mecamylamine (Mec; ganglionic blocker) and noradrenaline (NA; to restore vascular tone). The vehicle had no effects in the unblocked or ganglionic-blocked rats. Apelin decreased mean arterial pressure (MAP) and increased heart rate (HR), but it did not alter mean circulatory filling pressure in the unblocked rats. In the ganglionic-blocked rats, apelin did not alter heart rate but decreased mean arterial pressure and mean circulatory filling pressure. These results show that apelin is an arterial and venous dilator in vivo. The depressor effect of apelin is accompanied by tachycardia which is abolished by ganglion blockade.     

The apelin/APJ system as a therapeutic target in metabolic diseases

Introduction: Apelin, a bioactive peptide, is the endogenous ligand of APJ, a G protein-coupled receptor which is widely expressed in peripheral tissues and in the central nervous system. The apelin/APJ system is involved in the regulation of various physiological functions and is a therapeutic target in different pathologies; the development of APJ agonists and antagonists has thus increased.

Area covered: This review focuses on the in vitro and in vivo metabolic effects of apelin in physiological conditions and in the context of metabolic diseases.

Expert opinion: In experimental models, novel APJ agonists are efficient in vivo, to treat metabolic diseases and associated complications. However, more clinical trials are necessary to determine whether molecules that target APJ could become an alternative therapeutic strategy in the treatment of metabolic diseases and associated complications.     

Modulation of the apelin/APJ system in heart failure and atherosclerosis in man

Background and purpose:

The aim of this study was to determine whether the apelin/APJ system is altered in human cardiovascular disease by investigating whether the expression of apelin or its receptor is altered at the protein level.

Experimental approach:

Radioligand binding studies were used to determine apelin receptor density in human cardiac tissues. Apelin peptide levels in cardiovascular tissues were determined by radioimmunoassay. In vitro pharmacology was used to assess vasoactive properties of apelin in human coronary artery. Localization of apelin and its receptor in coronary artery was determined using immunohistochemistry.

Key results:

Apelin receptor density was significantly decreased in left ventricle from patients with dilated cardiomyopathy or ischaemic heart disease compared with controls, but apelin peptide levels remained unchanged. Apelin was up-regulated in human atherosclerotic coronary artery and this additional peptide localized to the plaque, colocalizing with markers for macrophages and smooth muscle cells. Apelin potently constricted human coronary artery.

Conclusions and implications:

We have detected changes in the apelin/APJ system in human diseased cardiac and vascular tissue. The decrease in receptor density in heart failure may limit the positive inotropic actions of apelin, contributing to contractile dysfunction. The contribution of the increased apelin levels in atherosclerotic coronary artery to disease progression remains to be determined. These data suggest a potential role for the apelin/APJ system in human cardiovascular disease.     

A pharmacokinetic study of CGS-8216, a benzodiazepine receptor ligand,in the rat

A rapid and sensitive method is described for the determination of CGS-8216 (a pyrazoloquinoline that displaces benzodiazepines from their binding sites in the brain but which reverses some of the behavioural actions of the benzodiazepines) in plasma using high-performance liquid chromatography with ultraviolet detection. CGS-9896 serves as the internal standard. The method is applied to a pharmacokinetic study of CGS-8216 in the rat. CGS-8216 was not detectable in plasma 24 h after a single IP administration of a 10 mg/kg dose. Animals treated with five once-daily injections of CGS-8216 had plasma concentrations 30 min after the final injection that were approximately four-times those observed 30 min after a single treatment. This suggests that caution must be used in the interpretation of results from experiments using multiple administrations of CGS-8216. The compound could not be detected in brain tissue at any of the time points studied.     

The hypolipidemic natural product guggulsterone is a promiscuous steroid receptor ligand

Guggulsterone (GS) is the active substance in guggulipid, an extract of the guggul tree, Commiphora mukul, used to treat a variety of disorders in humans, including dyslipidemia, obesity, and inflammation. The activity of GS has been suggested to be mediated by antagonism of the receptor for bile acids, the farnesoid X receptor (FXR). Here, we demonstrate that both stereoisomers of the plant sterol, (E)- and (Z)-GS, bind to the steroid receptors at a much higher affinity than to FXR. Both stereoisomers bind to the mineralocorticoid receptor (MR) with a Ki value of approximately 35 nM, which is greater than 100 times more potent than their affinity for FXR. Both (E)- and (Z)-GS also displayed high affinity for other steroid receptors, including the androgen (AR), glucocorticoid (GR), and progesterone receptors (PR) with Ki values ranging from 224 to 315 nM. In cell-based functional cotransfection assays, GSs behaved as antagonists of AR, GR, and MR, but as agonists of PR. Agonist activity was also demonstrated with estrogen receptor (ER) alpha; however, the potency was very low (EC50 > 5000 nM). In addition, GS displayed activity in functional assays in cell lines expressing endogenous AR, GR, ER, and PR. These data suggest that the variety of pharmacological effects exhibited by GS may be mediated by targeting several steroid receptors.     

beta-Cryptoxanthin, a novel natural RAR ligand, induces ATP-binding cassette transporters in macrophages

Despite its serious adverse effects, recent accumulating evidence suggests that a physiological retinoic acid receptor (RAR) agonist, all-trans retinoic acid (atRA), exhibits preventive effects on atherogenesis. Therefore, the present study was designed to explore novel natural RAR ligands with anti-atherogenic effects in order to identify and develop a drug without severe side effects. Among xanthophylls and carotenoids studied, beta-cryptoxanthin and lutein exhibited RAR ligand activity in yeast two-hybrid system that was found to be completely abolished by the RAR pan-antagonist LE540. Furthermore, these molecules can bind the RAR ligand-binding domain in the CoA-BAP system but not RXR ligand-binding domain. These results indicate that both beta-cryptoxanthin and lutein serve as ligands for RAR, but not RXR, although their binding affinity was three orders of magnitude lower than that of atRA. Additionally, when applied to macrophages, beta-cryptoxanthin indeed was found to induce the ATP-binding cassette transporter A1 (ABCA1) and ABCG1 mRNAs, which exert anti-atherosclerotic effects by preventing cholesteryl ester accumulation in macrophages. The induction of ABCA1 proteins by beta-cryptoxanthin as well as atRA was abrogated by LE540. In summary, beta-cryptoxanthin appears to be more an efficient provitamin A source than other carotenoids and xanthophylls including beta-carotene, since beta-cryptoxanthin can act not only as a RAR agonist but also a source of vitamin A. Taking into account that the pharmacodynamics difference between beta-cryptoxanthin and atRA, beta-cryptoxanthin appears to exert beneficial effects on atherogenesis through RAR activation in the manner different from atRA.     

Urocontrin, a novel UT receptor ligand with a unique pharmacological profile

In recent years, several studies have demonstrated that urotensin II (UII) and urotensin II-related peptide (URP) can exhibit differential biological activity. So far, known antagonists of the urotensin II receptor (UT) are of limited usefulness for investigating the specific pathophysiological role of UII or URP. Therefore, identification of new compounds able to discriminate UII- and URP-associated biological activities is crucially needed. In the present study, we report preliminary data regarding the pharmacological properties of a novel UT ligand termed urocontrin, i.e. [Bip(4)]URP, that is able to reduce the ex vivo efficacy of hUII- but not URP-induced vasoconstriction in rat aortic rings. In vivo studies support the pharmacological profile described above. Although urocontrin exert some residual agonist activity, this compound should be useful for the rational design of potent molecules that would allow discriminating specific biological action mediated by UII or URP.     

Melanocortin-4 receptor antagonists as potential therapeutics in the treatment of cachexia

The melanocortin-4 (MC4) receptor subtype plays a pivotal role in body weight regulation. Knock-out or mutation of MC4 receptors in animals or humans leads to severe obesity and acute or sub-acute antagonism of central MC4 receptors produces an increase in food intake and a decrease in metabolism. Knock-out or antagonism of MC4 receptors in animal models of cachexia leads to a protection from anorexia and the loss of both lean and fat body mass, suggesting that an MC4 antagonist may be beneficial in wasting diseases, which are poorly treated by available therapies. Considerable progress has been made in the discovery of non-peptide antagonists with high affinity and selectivity for MC4 receptors. Optimization of these compounds has produced molecules that are active upon systemic administration and are effective in protecting against cachectic symptoms in animal models of tumor-induced wasting. Further development of such compounds is greatly anticipated as a potential means to combat the cachexia that results from chronic diseases such as cancer, AIDS, renal failure, liver failure, congestive heart failure and lung disease.     

125I]thienylphencyclidine, a novel ligand for the NMDA receptor.

We have monitored the binding of [125I]thienylphencyclidine ([125I]TCP), a novel high affinity radioiodinated ligand that specifically recognizes the NMDA (N-methyl-D-aspartate) receptor in rat brain membranes. [125I]TCP binds with an affinity of about 30 nM, and recognizes a similar number of binding sites to previously employed ligands for this receptor. [125I]TCP binding is characterized by slow association and dissociation rates, and the latter can be modified by the addition of Mg2+ or Zn2+, as previously described for [3H]dizocilpine ([3H]MK801). Other phencyclidine-like ligands displaced [125I]TCP binding with the order of potency dizocilpine greater than thienylphencyclidine greater than ITCP greater than phencyclidine greater than ketamine. The binding of [125I]TCP was also increased by NMDA and glycine-site agonists and inhibited by antagonists of these sites. Surprisingly, however, the polyamines spermidine and spermine did not increase [125I]TCP, even though the polyamine antagonist arcaine was an effective inhibitor of binding. These results show that [125I]TCP is a useful ligand for the NMDA receptor complex that binds to the receptor in a manner that is qualitatively distinct from previously described ligands.     

高盐上调压力超负荷大鼠的脑内APJ受体表达

目的观察高盐对压力超负荷大鼠的交感神经活性和脑内Apelin和APJ系统的影响。方法♂SD大鼠,在肾动脉分支上方部分结扎腹主动脉,建立主动脉缩窄大鼠模型。术后4周,分别给予含8%Na Cl的高盐饲料或含0.3%Na Cl的常规饲料,喂养1周后,记录各组大鼠的血流动力学变化;ELISA分析24 h尿去甲肾上腺素(NE)水平;real-time RCR和Western blot方法观察室旁核Apelin和APJ的表达变化。另一组实验中,在给予高盐饲料的同时使用Alzet微渗透泵侧脑室灌流ML221或benzamil,观察24 h尿NE水平和心脏指数(HW/BW)变化。此外,在正常SD大鼠侧脑室灌流高钠脑脊液1周后,观察大鼠血压、24 h尿NE和APJ表达水平的变化。结果压力超负荷大鼠给予高盐饮食1周后,平均动脉血压(MAP)、心率(HR)、左室收缩末期压(LVESP)和24 h尿NE水平,以及室旁核的APJ mRNA和蛋白表达均较对照明显增加(P<0.05);而假手术组大鼠在进食高盐饲料后,血流动力学、24 h尿NE水平和APJ表达均未见明显改变。在侧脑室分别灌注APJ受体拮抗剂ML221或ENa C拮抗剂benzamil后,可以明显抑制高盐引发的压力超负荷大鼠24h尿NE水平和HW/BW比值变化(P<0.01)。在正常血压大鼠侧脑室灌注高Na+脑脊液1周后,也可出现脑内APJ表达上调以及MAP、HR和尿NE水平升高(P<0.01)。结论高盐可以增加压力超负荷大鼠模型的交感神经活性,而该效应与脑内的APJ受体表达上调和Na+敏感性增加密切相关。APJ受体可能是脑内盐敏感性形成的重要调控靶点。     

Neurochemical characterization of dopaminergic effects of opipramol, a potent sigma receptor ligand, in vivo

Opipramol, a tricyclic antidepressant drug, potently interacted with sigma recognition sites labelled by [³H](+)-3-(3-hydroxyphenyl)N-(1-propyl)piperidine ([³H](+)-3-PPP) with a K_i value of 50±8nM and with minimal affinity for phencyclidine receptors (K_i > 30,000 nM). Opipramol potently increased the metabolism of dopamine in the striatum, olfactory tubercle and pyriform cortex of the rat and increased the release of dopamine from the striatum of the mouse, as measured by increases in the levels of 3-methoxytyramine in vivo. Opipramol increased plasma prolactin in the rat, only at a dose as large as 50 mg/kg dose. Irreversible inactivation of dopamine receptors by EEDQ (N-ethoxycarbonyl-2-ethoxy-1, 2-dihydroquinoline) did not affect the opipramol-induced increases in levels of dihydroxyphenyl- acetic acid (DOPAC) in the striatum of the rat, indicating a predominant role of activation of sigma receptors in the dopaminergic effects of opipramol, However, pretreatment with the putative sigma ligand, rimcazole, markedly potentiated the ability of opipramol to increase the metabolism of release of DA in the striatum of the mouse in vivo. These results suggest that rimcazole and opipramol interact at two distinct receptors, the pharmacological significance of which is yet to be elucidated.     

Imiquimod, a toll-like receptor 7 ligand, inhibits airway remodelling in a murine model of chronic asthma

1. Imiquimod, a synthetic Toll-like receptor (TLR) 7 ligand, has been shown to attenuate airway inflammation and airway hyperresponsiveness (AHR) in acute murine models of allergic asthma. In the present study, we investigated the effect of imiquimod on allergen-induced airway remodelling in chronic experimental asthma. 2. Ovalbumin (OVA)-sensitized mice were chronically challenged with aerosolized OVA for 8 weeks. Some mice were exposed to an aerosol of 0.15% imiquimod daily during the period of OVA challenge. Twenty-four hours after the last OVA challenge, mice were evaluated for the development of airway inflammation, AHR and airway remodelling. The levels of total serum IgE and Th2 cytokines (interleukin (IL)-4, IL-5 and IL-13) in bronchoalveolar lavage fluid (BALF) and the expression of transforming growth factor (TGF)-beta1 protein in lungs were measured by ELISA and immunohistochemistry, respectively. 3. The results demonstrated that imiquimod significantly inhibited chronic inflammation, persistent AHR and airway remodelling in chronic experimental asthma. In addition, imiquimod reduced levels of total serum IgE and BALF Th2 cytokines and diminished expression of TGF-beta1 in remodelled airways. 4. In summary, the results of the present study indicate that imiquimod may attenuate the progression of airway inflammation and remodelling, providing potential in the treatment of asthma.     

A PET study of D1-like dopamine receptor ligand binding

Rationale: Several positron emission tomography (PET) studies have shown that radioligand binding to D₂-like dopamine receptors competes with endogenous dopamine. Objective: The purpose of this PET study was to examine the effect of amphetamine and reserpine on D₁-like dopamine receptor binding. Methods: Three Cynomolgus monkeys were examined with the radioligands [¹¹C]SCH 23390 or [¹¹C]NNC 112 at baseline condition and after pretreatment with amphetamine (2 mg/kg IV). The B/F values (binding potential) in the striatum and the neocortex were calculated at transient equilibrium. In two monkeys, the effect of the long-lasting dopamine depletion after reserpine (1 mg/kg IV) was followed by a repeated Scatchard procedure in up to 77 days after drug administration. The Scatchard analysis was based on two PET measurements with high and low specific radioactivity and allowed the calculation of D₁-like dopamine receptor density (B_max) and apparent affinity (K_D ^app). Results: The effect of amphetamine on the B/F values was between –14 and 6%. These changes can be considered as within the range of the test-retest reliability. Thus, there was no evident effect of amphetamine-induced dopamine release on D₁-like dopamine receptor binding. Five hours after reserpine administration, there was no change in B_max or K_D ^app. At 3, 23, and 28 days after reserpine administration, the Scatchard analyses indicated a 13–20% reduction in B_max without any evident change in K_D ^app in both the striatum and the neocortex. Conclusions: The lack of evident effects of amphetamine and reserpine on D₁-like dopamine receptor binding is markedly different from the 20% amphetamine-induced decrease and 50% reserpine-induced increase that has been consistently reported for D₂-like dopamine receptor binding. The data indicated that D₁-like dopamine receptor occupancy of endogenous dopamine is low at physiological condition. It is thus unlikely that D₁-like dopamine receptor radioligands can be used to measure changes in the concentration of endogenous dopamine. Received: 8 March 1999 / Final version: 12 May 1999     

Discovery of a highly active ligand of human pregnane x receptor: a case study from pharmacophore modeling and virtual screening to "in vivo" biological activity

The human pregnane X receptor (hPXR) is a nuclear receptor that regulates the expression of phase I and II drug-metabolizing enzymes as well as that of drug transporters. In addition, this receptor plays a critical role in cholesterol homeostasis and in protecting tissues from potentially toxic endobiotics. hPXR is activated by a broad spectrum of low-affinity compounds including xenobiotics and endobiotics such as bile acids and their precursors. Crystallographic studies revealed a ligand binding domain (LBD) with a large and conformable binding pocket that is likely to contribute to the ability of hPXR to respond to compounds of varying size and shape. Here, we describe an in silico method that allowed the identification of nine novel hPXR agonists. We further characterize the compound 1-(2-chlorophenyl)-N-[1-(1-phenylethyl)-1H-benzimidazol-5-yl]methanesulfonamide (C2BA-4), a methanesulfonamide that activates PXR specifically and more potently than does the reference compound 4-[2,2-bis(diethoxyphosphoryl)ethenyl]-2,6-ditert-butyl-phenol (SR12813) in our stable cell line expressing a Gal4-PXR and a GAL4 driven luciferase reporter gene. Furthermore treatment of primary human hepatocytes with C2BA-4 results in a marked induction of the mRNA expression of hPXR target genes, such as cytochromes P450 3A4 and 2B6. Finally, C2BA-4 is also able to induce hPXR-mediated in vivo luciferase expression in HGPXR stable bioluminescent cells implanted in mice. The study suggests new directions for the rational design of selective hPXR agonists and antagonists.