Anna Lorenzen

Reduction of postischemic leukocyte-endothelium interaction by adenosine via A2 receptor

SummaryThe adhesion of leukocytes to the endothelium of postcapillary venules hallmarks a key event in ischemia-reperfusion injury. Adenosine has been shown to protect from postischemic reperfusion injury, presumably through inhibition of postischemic leukocyte-endothelial interaction. This study was performed to investigate in vivo by which receptors the effect of adenosine on postischemic leukocyte-endothelium interaction is mediated.The hamster dorsal skinfold model and fluorescence microscopy were used for intravital investigation of red cell velocity, vessel diameter, and leukocyte-endothelium interaction in postcapillary venules of a thin striated skin muscle. Leukocytes were stained in vivo with acridine orange (0.5 mg kg−1 min−1 i.v.). Parameters were assessed prior to induction of 4 h ischemia to the muscle tissue and 0.5 h, 2 h, and 24 h after reperfusion. Adenosine, the adenosine A1-selective agonist 2-chloro-N6-cyclopentyladenosine (CCPA), the A2-selective agonist CGS 21,680, the non-selective adenosine receptor antagonist xanthine amine congener (XAC), and the adenosine uptake blocker S-(p-nitrobenzyl)-6-thioinosine (NBTI) were infused via jugular vein starting 15 min prior to release of ischemia until 0.5 h after reperfusion.Adenosine and CGS 21,689 significantly reduced postischemic leukocyte-endothelium interaction 0.5 h after reperfusion (p<0.01), while no inhibitory effect was observed with CCPA. Coadministration of XAC blocked the inhibitory effects of adenosine. Infusion of NBTI alone effectively decreased postischemic leukocyte-endothelium interaction.These findings indicate that adenosine reduces postischemic leukocyte-endothelium interaction via A2 receptor and suggest a protective role of endogenous adenosine during ischemia-reperfusion.

G protein-coupled receptor for nicotinic acid in mouse macrophages.

The use of the HDL-elevating drug nicotinic acid in the treatment and prevention of atherosclerotic disease is limited by the frequent induction of skin flushing. The therapeutic effects of nicotinic acid are attributed to inhibition of lipolysis in adipose tissue via a G protein-coupled receptor, whereas the mechanism of flush induction by release of prostaglandin D(2) from macrophages is not understood. In this study, we investigated if macrophages contain nicotinic acid receptors. Specific guanine nucleotide sensitive binding sites for [(3)H]nicotinic acid were detected in membranes from mouse RAW 264.7 macrophages. Nicotinic acid and related heterocycles stimulated activation of pertussis toxin-sensitive G proteins. The rank orders of potency in macrophage membranes were identical for inhibition of [(3)H]nicotinic acid binding and G protein activation, and were pharmacologically indistinguishable from that of the G protein-coupled nicotinic acid receptor in spleen membranes. These results indicate that the effects of nicotinic acid on macrophages, spleen and probably adipocytes are mediated via an identical, unique G protein-coupled receptor.

Differences in the order of potency for agonists but not antagonists at human and rat adenosine A2A receptors

To examine possible species differences in pharmacology, rat adenosine A2A receptors were studied in PC12 (pheochromocytoma) cells, and human receptors in Chinese hamster ovary (CHO) cells transfected with the cloned human A2A receptor cDNA. Using [3H]-5-amino-7(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo [1,5-c]pyrimidine ([3H]-SCH 58261) as radioligand, the estimated Bmax (maximal binding) was 538 and 2085 fmol/mg in CHO and PC12 cells, respectively. The Kd (dissociation constant) values for [3H]-SCH 58261 were 1.05 and 5.6 nM in the two cell types, respectively. The order of potency of antagonists and most agonists was the same in both cell types, but 2-phenylaminoadenosine and 2-chloroadenosine were relatively less potent in PC12 cells than in CHO cells. In the functional assay, using cyclic AMP accumulation, all agonists tested were more potent in CHO than in PC12 cells, but this could not be readily explained by differences in adenylyl cyclase or in the expression of G proteins. As in the case of binding, the relative agonist potencies were similar for most compounds, but 2-phenylaminoadenosine and 2-chloroadenosine were more potent at human A2A receptors in CHO cells than predicted from the data obtained on rat A2A receptors in PC12 cells. Antagonists were approximately equipotent in the two cells. These results show that, despite only small differences in amino acid sequences and no difference in antagonist pharmacology, the relative order of potency of receptor agonists can differ between species homologues of the adenosine A2A receptor.

Physiology and pharmacology of natural and synthetic nonadenine-based purines in the nervous system

Like their adenine‐based counterparts, increasing evidence implicates extracellular nonadenine‐based purines such as guanosine and GTP as trophic effector molecules, affecting the growth and differentiation of cells in the nervous system. The extracellular concentration of guanosine is higher than that of adenosine, both in physiological and pathological conditions. Extracellular guanosine and GTP stimulate the astrocyte cell division, apparently through enhancing release of their adenine‐based counterparts, which act in an autocrine fashion. Guanosine and GTP also stimulate the synthesis by astrocytes of several neurotrophic factors, e.g., NGF, and bFGF, and the release of NGF and S100β. As well, guanosine and GTP enhance the differentiation of PC12 cells and hippocampal neurons in vitro. Their action on PC12 cells is associated with the early synthesis of adenotin‐1, a chaperone protein. A hypoxanthine analog, AIT‐082, has similar activity on PC12 cells and neurons to guanosine, but is not metabolized. It enhances memory in both old memory‐deficient mice and in young mice, stimulates neurotrophic factor synthesis in astrocytes in vitro and in brain in vivo, and protects hippocampal neurons in vitro from a “dying‐back” neuropathy caused by brief exposure to high concentrations of glutamate. Given systemically, it protects neurons from NMDA‐induced toxicity. Its neuroprotective effects may partly be related to its ability to stimulate release of NGF from astrocytes. Although AIT‐082 enhances NGF synthesis, unlike exogenously administered NGF it does not produce hyperalgesia. AIT‐082 may prove useful in the treatment of Alzheimers disease, for which it is in Phase II trials, and also in the treatment of acute neuronal injuries due, e.g., to trauma and stroke. Drug Dev. Res. 45:356–372, 1998.

Thermodynamically distinct high and low affinity states of the A1 adenosine receptor induced by G protein coupling and guanine nucleotide ligation states of G proteins

The influence of the receptor‐G protein coupling state and the guanine nucleotide ligation state of the G protein on the binding mechanism of A1 adenosine receptor ligands has been investigated in [3H]‐1,3‐dipropyl‐8‐cyclopentylxanthine ([3H]‐DPCPX) binding studies in rat brain membranes. Thermodynamic parameters of binding of A1 adenosine receptor ligands of different intrinsic activities were determined in the absence or presence of GDP and compared to the binding mechanism after receptor‐G protein uncoupling. In agreement with previous studies, it was found that xanthine and non‐xanthine antagonists showed an enthalpy‐ or enthalpy‐ and entropy‐driven binding mechanism under all conditions. In contrast to antagonists, the binding mechanism of agonists was strongly affected by the G protein coupling state or the absence or presence of guanine nucleotides. Binding of full and partial agonists to the high‐affinity state of the A1 receptor was entropy‐driven in the absence of GDP, and a good correlation between intrinsic activities and the contribution of entropy was observed. In the absence of GDP, binding of full and partial agonists and antagonists to the high affinity state of the receptor was thermodynamically discriminated. In contrast, no such discrimination was found in the presence of GDP. The binding mechanism of agonists to the low‐affinity state of the receptor was identical to that of antagonists only after uncoupling of the receptor from G proteins by pretreatment with N‐ethylmaleimide or guanosine‐5′‐(γ‐thio)‐triphosphate (GTPγS). These results indicate the existence of two thermodynamically distinct high‐ and low‐affinity states of the A1 adenosine receptor.

Identification of a novel high affinity adenosine binding protein from bovine striatum.

SummaryIn solubilized extracts from bovine striatal membranes three different binding sites for 5′-N-ethylcarboxamidoadenosine ([3H]NECA) were observed after separation of the extract by gel filtration on Sepharose CL-6B. The first peak was eluted in the void volume and contained the AZ adenosine receptor. In the second peak, [3H]NECA binding sites were eluted with a pharmacological profile characteristic of adenotin, a low affinity non-receptor adenosine binding protein. The third peak represented approximately 50% of the [3H]NECA binding activity. This site bound [3H]NECA in a reversible and saturable manner with KD of 17 nmol/l and a binding capacity of 11.3 pmol/mg protein. In competition experiments, adenosine, NECA, NAD, nnosine, 5′-AMP and S-adenosyl-L-homocysteine were the most potent ligands. In contrast to adenosine receptors, this site did neither bind adenosine receptor antagonists nor the A2 selective agonist CGS 21,680 (2-[p-(2-carboxyethyl)phenethylamino]5′-N-ethylcarboxamidoadenosine). These results suggest the existence of a novel high affinity binding site for adenosine of unknown function in bovine striatum.

Characterization of membrane-bound and solubilized high-affinity binding sites for 5'-N-ethylcarboxamido[3H]adenosine from bovine cerebral cortex.

Abstract: A high‐affinity binding site for 5′‐N‐ethylcarboxamido[3H]adenosine ([3H]NECA) from bovine cerebral cortex has been characterized in its membrane‐bound and solubilized state after gel filtration on Sepharose CL‐6B. For detection of this site in membranes, it was necessary to remove metabolites with high affinities for this site enzymatically, e.g., adenosine by addition of adenosine deaminase and inosine by addition of nucleoside phosphorylase. The pore‐forming peptide antibiotic alamethicin further enhanced binding of [3H]NECA to this site in membranes. In contrast to adenosine receptors and the adenotin‐like low‐affinity binding protein, this novel site was extremely sensitive against treatment with the sulfhydryl alkylating agent N‐ethylmaleimide. In competition experiments, this site could be differentiated from adenosine receptors by its high affinity for adenine nucleotides and its lack of affinity for adenosine receptor antagonists. Inosine and its derivative S‐(4‐nitrobenzyl)‐6‐thioinosine were relatively potent ligands with Ki values in the high nano‐ and low micromolar range, respectively. We conclude that the high‐affinity NECA binding site described previously in bovine striatum is not exclusively located in the striatum, but can also be detected in membrane preparations and soluble extracts of bovine brain cortex.

Inverse agonism at adenosine A1 receptors

Abstract Inverse agonism at adenosine A1 receptors was studied in a variety of experimental set-ups. As a read-out, the binding of [35S]GTPγS to membranes of either CHO or COS-7 cells expressing human adenosine A1 receptors was used. When wild-type receptors were studied, inverse agonism could only be detected at higher levels of receptor expression. However, receptors fused with (mutated) α-subunits of Gi proteins facilitated the detection of inverse agonism, which could be studied in these systems at lower levels of expression. Ligands previously all classified as antagonists behaved differently, even within series of chemical homologues. For instance, in the xanthine class, theophylline and caffeine were neutral antagonists, whereas another xanthine, 1,3-dipropyl,8-cyclopentylxanthine (DPCPX), invariably emerged as a high-affinity inverse agonist. During our investigations, it became apparent that the neutral antagonists identified so far had all modest affinity for the receptor. Therefore, we started a program aimed at the design and synthesis of neutral antagonists with higher affinity. N-0840, N6-cyclopentyl-9-methyladenine, another neutral antagonist, was used as a lead structure. We succeeded in synthesizing a new series of C8-substituted N-0840 derivatives, many of which had higher affinity than the parent compound. Interestingly, some of the compounds turned out to be inverse agonists, whereas others maintained their neutral antagonistic character.

Human choriogonadotropin entrapped into liposomes: characterization, biologic effects and interaction with purified mouse Leydig cells in vitro

Liposomes containing human choriogonadotropin (hCG) were prepared from phosphatidylserine by the ether injection method. hCG adsorbed to the outer surface of the liposomes (77% of total liposome-associated hCG) was removed by proteolytic digestion with subtilisin. hCG-containing liposomes digested and not digested with subtilisin stimulated testosterone biosynthesis by Leydig cells in a dose-dependent way; both preparations had identical biologic activities (32% of the activity of free, not liposome-associated hCG) when equal doses of liposome-associated hCG were applied. The onset of stimulation was delayed when compared to the action of free hCG. Liposomes without hCG did not stimulate testosterone biosynthesis. Association of liposomes with Leydig cells was determined by measurement of transfer of radioactive label from liposomes to Leydig cells. The association was not mediated by the hormone receptor. hCG entrapped in liposomes was incorporated by Leydig cells and translated to the cellular surface. This process was impaired by colchicine (10(-5) M). hCG translocated to the external surface of the cell membrane contained a modified alpha-subunit (Mr 16,200 instead of 20,600) which was not detected in unentrapped hCG bound to Leydig cells. We suggest that liposomally entrapped hCG is taken up by Leydig cells and re-exported to the cell membrane by a mechanism resembling retroendocytosis.