Ingeborg van der Ploeg

Evidence for functionally important adenosine A2a receptors in the rat hippocampus

Adenosine A2a receptors are not confined to dopamine-rich areas of the brain, since thermocycling analysis shows that adenosine A2a receptor mRNA is expressed also in the hippocampus (CA1, CA3 and dentate gyrus) and cerebral cortex. The expression of A2a mRNA in three main areas of the hippocampus was confirmed by in situ hybridization; A2a mRNA expression was mainly localized in the pyramidal and granular cells, the same hippocampal regions that showed adenosine A1 receptor mRNA expression. Receptor autoradiographic studies with [3H]CGS 21680 (30 nM), a selective adenosine A2a receptor agonist, showed specific binding sites in the hippocampus. The density of [3H]CGS 21680 binding was greatest in the stratum radiatum of the CA1 area, followed by the stratum oriens of the cornu Ammonis, stratum radiatum of the CA3 are and supra-granular layer of the dentate gyrus. This anatomical distribution of [3H]CGS 21680 binding was similar to the pattern of [3H]CHA binding in the hippocampus. Electrophysiological studies in the Schaffer fibers/CA1 pyramids showed that upon activation of the A2a receptors with CGS 21680 (10 nM) the ability of the adenosine A1 receptor agonist, CPA, to inhibit neuronal activity was significantly attenuated. These results show functionally important co-expression and co-localization of adenosine A2a and A1 receptors in the hippocampus. The results also suggest that adenosine A2a receptor-mediated neuromodulation is not confined to the basal ganglia, but is more widespread throughout the nervous system.

Effect of long term caffeine treatment on A1 and A2 adenosine receptor binding and on mRNA levels in rat brain

SummaryThe effect of long-term oral treatment with caffeine on A1 and A2 receptors in the rat brain was studied. Caffeine was added to the drinking water and the animals were sacrificed after a 12 day treatment period. The plasma caffeine concentration was close to 100 μM. A1 receptors were studied using quantitative autoradiography with [3H]cyclohexyladenosine (CHA). Caffeine treatment increased the number of A1 receptors in the CA3 subfield of the hippocampus from 337 to 393 fmol/mg with no change in KD (0.692 vs. 0.675 nM). A1 mRNA was measured using Northern blots and quantitative in situ hybridization. There was no increase in A1 mRNA. A2a receptors, located in dopamine rich regions of the rat brain, were studied with quantitative autoradiography using [3H]CGS 21680 as the ligand, and the A2a mRNA was determined using quantitative in situ hybridization. Caffeine treatment produced no significant change in either receptor number or mRNA, even though the apparent Bmax tended to increase from 322±8 to 352±8 fmol/mg. The results show that treatment with caffeine in a dose that causes tolerance to several effects of caffeine and increases some effects of adenosine analogues increases the number of A1 receptors without any change in A1 mRNA, suggesting that the adaptive changes are at a post-translational level. There were no significant changes in A2 receptors indicating that the two types are regulated differently and/or that the amount of endogenous agonist is sufficient to regulate A1, but not A2 receptors.

Adenosine A2A receptors mediate the inhibitory effect of adenosine on formyl-Met-Leu-Phe-stimulated respiratory burst in neutrophil leucocytes

AbstractUsing the reverse transcription polymerase chain reaction (RT PCR) we found that human neutrophils express mRNA for both A2A and A2B adenosine receptors, and using selective adenosine receptor agonists and antagonists we have characterized the type of adenosine receptor mediating inhibition of formyl-Met-Leu-Phe (fMLP)-induced oxidative burst. The order of potency of agonists was 5′-N-ethyl-carboxamidoadenosine (NECA) >2-phenylaminoadenosine>2-[p-(2-carbonyl-ethyl)-phenyl-ethylamino]-5′-N-ethylcarboxamidoadenosine (CGS 21680)>adenosine> N6-cyclopentyladenosine. This agrees with the agonist potency at human A2A receptors. The effect of adenosine was antagonized by 30 μM theophylline>caffeine = paraxanthine, i.e. concentrations close to those occurring in plasma after consumption of caffeine-containing beverages. The effect of NECA was unaltered by the A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine, but inhibited by the A2A receptor selective antagonists 4-amino-8-chloro-1-phenyl-[1,2,4]-triazolo-[4,3-a]quinoxaline (CP 66,713), 1,3-dipropyl-8-(3,4-dimethoxystyryl)-7-methylxanthine (KF 17387) and 8-(3-chlorostyryl)caffeine as well as by the non-selective, non-xanthine antagonist 5-amino-9-chloro-2-(2-furyl)-[1,2,4]-triazolo-[1,5-c]quinazoline methane sulphonate (CGS 15943). The adenosine receptor mediated responses were antagonized by the protein kinase A blocker Rp-cyclic adenosine 3′,5′-phosphorothioate (Rp-cAMP). In conclusion, the adenosine-induced inhibition of neutrophil activation is mediated by adenosine A2A receptors.

Not only Th2 cells but also Th1 and Th0 cells express CD30 after activation.

To investigate whether the CD30 molecule, expressed only by a minority of T and B cells, defines a subtype of T helper cells, Pityrosporum orbiculare‐specific CD4+ T cell clones were assessed for CD30 protein and gene expression. The clones were defined as Th1, Th0, and Th2 according to their cytokine mRNA profile detected by reverse transcription PCR (RT‐PCR). The kinetics of CD30 expression after OKT3 (anti‐CD3) stimulation was analyzed by flow cytometry, immunocytochemistry, and RT‐PCR. OKT3 activation induced a high expression of CD30 in cells of both Th1 and Th0 as well as Th2 type after 1‐3 days. A difference between the clones was noted in that the Th2 clones remained highly positive in CD30 expression, whereas expression in the other clones started to decline from day 3. These data indicate that CD30 is expressed in activated CD4+ T cells of all three subtypes, and that the expression is sustained in Th2 cells.

Neurotensin decreases the affinity of dopamine D2 agonist binding by a G protein-independent mechanism.

Abstract: To examine whether GTP‐binding proteins (G proteins) mediate the ability of neurotensin to lower the affinity of dopamine D2 agonist binding, the modulation by neurotensin in vitro of N‐[3H]propylnorapotnorphine ([3H]NPA) binding was investigated following pretreatment with pertussis toxin and N‐ethylmaleimide in rat neostriatal membranes. Preincubation with N‐ethylmaleimide (100 μM) markedly inhibited pertussis toxin‐induced back‐ADP ribosylation of three proteins with apparent molecular masses of 41, 40, and 39 kDa, respectively. This inhibition was prevented by adding dithiothreitol (250 μM) during the preincubation. N‐Ethylmaleimide increased the KD (180 ± 30%) and decreased the Bmax (−31 ± 9%) of [3H]NPA binding sites but did not affect the binding properties of the selective D2 antagonist [3H]raclopride. N‐Ethylmaleimide pretreatment did not affect the neurotensin (3 nM)‐induced increase in the KD of [3H]NPA binding sites. Pertussin toxin treatment in vivo and in vitro was similarly ineffective. In conclusion, the present study indicates that neurotensin modulation of D2 agonist binding in neostriatal membranes is not mediated by G proteins.

FUNCTIONAL CHARACTERIZATION OF ADENOSINE A2 RECEPTORS IN JURKAT CELLS AND PC12 CELLS USING ADENOSINE RECEPTOR AGONISTS

The effect of several adenosine analogues on cyclic AMP accumulation was examined in the rat phaeochromocytoma cell PC12 and in the human T-cell leukaemia cell Jurkat, selected as prototypes of cells predominantly expressing adenosine A2A or A2B receptors. Using the reverse transcription-polymerase chain reaction it was, however, demonstrated that the Jurkat cell and the PC12 cell express both A2A and A2B receptor mRNA, albeit in different relative proportions. In PC12 cells the concentration required for half-maximal response (EC50) for the full agonist 5′-N-ethyl-car-boxamidoadenosine (NECA) was 30 times lower than in Jurkat cells. There was no significant difference in the pA2 for the antagonist 5-amino-9-chloro-2-(2-furanyl)1,2,4-triazolo(1,5-C)quinazolinemonomethanesulphon-ate (CGS 15943) between the two cell types. In the presence of forskolin (1 μM in PC12 cells; 10 μM in Jurkat cells) the EC50 value for NECA was reduced two-to sixfold. Forskolin also increased the maximal cAMP accumulation twofold in PC12 cells and sevenfold in Jurkat cells. A series of 2-substituted adenosine analogues CV 1808 (2-phenylamino adenosine), CV 1674 [2-(4-methoxyphenyl)adenosine], CGS 21680 {2-[p-(2-carbonylethyl)phenylethylamino]-5′-N-ethyl-carboxamido adenosine}, and four 2-substituted isoguanosines, SHA 40 [2-(2-phenylethoxy)adenosine; PEA], SHA 91 [2-(2-cyclohexylethoxy)adenosine; CEA], SHA 118 {2-[2-(p-methylphenyl)ethoxy]adenosine; MPEA}, and SHA 125 (2-hexyloxyadenosine; HOA), all raised CAMP accumulation in PC12 cells, but had minimal or no effect in Jurkat cells. In the PC12 cells the addition of forskolin (1 μM) reduced the EC50 by a factor of 2 (CV 1808) to 12 (SHA 125). In Jurkat cells all the analogues gave a significant, but submaximal, cAMP response in the presence of forskolin (10 μM), but they were essentially inactive in its absence. The results show that a series of 2-substituted adenosine analogues can be used to discriminate between A2A and A2B receptors. The two receptor subtypes appear to coexist, even in clonal cells selected for typical pharmacology. A2 receptor pharmacology can therefore be complex.

In vivo pertussis toxin treatment attenuates some, but not all, adenosine A1 effects in slices of the rat hippocampus ☆

In order to examine the involvement of G-proteins in mediating the different effects of adenosine A1-receptor stimulation in rat hippocampus we injected pertussis toxin (PTX) intraventricularly close to the hippocampus and examined its effect in slices 48-60 h later. The in vivo PTX treatment caused a partial (50 +/- 5%) inhibition of the [32P]ADP ribosylation produced by PTX added together with [32P]NAD in vitro. Such PTX treatment eliminated the electrophysiologically determined gamma-amino-n-butyric acid (GABA)B receptor response in the hippocampal CA1 region, but GABAA effects were unaffected. The adenosine (50 microM)-mediated hyperpolarization and decrease in input resistance as well as the adenosine-mediated inhibition of low calcium-induced bursting in pyramidal CA1 neurons were virtually abolished. The same was true for the decrease in [3H]cyclic AMP accumulation that is produced by the adenosine analogue R-N6-phenylisopropyl adenosine (R-PIA) in forskolin-treated hippocampal slices. As far as modulation of transmitter release was concerned, the R-PIA (1 microM)-induced inhibition of release of both [3H]noradrenaline (NA) and [3H]acetylcholine (ACh) evoked by field stimulation in hippocampal slices was affected hardly or not at all by pertussis toxin treatment. The inhibitory effect of adenosine on field excitatory postsynaptic potential (EPSP)s evoked in the CA1 region was unaltered by PTX pretreatment. The present results show that in vivo pertussis toxin treatment can inhibit some but not all A1-adenosine-receptor effects. This strongly suggests that closely similar A1 receptors might be coupled to G-proteins that differ in their sensitivity to PTX treatment.

cDNA analysis of the mite allergen Lep d 1 identifies two different isoallergens and variants.

For the first time the complete cDNA encoding two isoallergens of the non‐pyroglyphid dust mite Lepidoglyphus destructor, Lep d 1, allergen has been sequenced. In addition, one of the isoallergens was found to have two variants. Oligonucleotides were designed from known amino acid sequences. The cDNA sequences were obtained by hybridizing these primers to mRNA and enhancement by the RT‐PCR technique. To obtain the different complete encoding cDNA sequences and eliminate heteroduplex artifacts, we performed PCR + 1 reactions. Comparison of the amino acid sequence of the allergen shows leader sequences of 16 amino acids for both isoforms.

Inhibition of VEGF secretion and experimental choroidal neovascularization by picropodophyllin (PPP), an inhibitor of the insulin-like growth factor-1 receptor.

INTRODUCTION Choroidal neovascularization (CNV) is a debilitating complication of age-related macular degeneration (AMD) and a leading cause of vision loss. Along with other angiogenic factors such as vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF)-1 and its receptor, IGF-1R, have been implicated in CNV. PURPOSE A prior study has shown that the cyclolignan picropodophyllin (PPP) efficiently blocks the insulin-like growth factor-1 receptor (IGF-1R) activity and causes cell death in uveal melanoma cell lines and in an in vivo model. In this study we investigated the effect of PPP on VEGF expression, both in vitro and in vivo, and whether this effect has antiangiogenic consequences in a murine CNV model. METHODS C57BL/6J mice with laser-induced CNVs were treated with PPP. Effects on CNV area were assayed by image analysis. VEGF levels in the choroid and retinal pigment epithelial cells (ARPE-19) were measured by Western blot or ELISA. Transcriptional activation of the VEGF promoter was determined by luciferase reporter gene assay. RESULTS Mice treated with PPP, administered intraperitoneally or orally, showed a 22% to 32% (P = 0.002) decrease in CNV area. Furthermore, VEGF levels in the choroid were significantly reduced. In cultured ARPE-19 cells, IGF-1 was shown to increase VEGF secretion. This increase was completely blocked by PPP. PPP reduced the level of transcriptional activity of the VEGF promoter. CONCLUSIONS PPP reduces IGF-1-dependent VEGF expression and CNV in vivo. Accordingly, IGF-1R inhibitors may be useful tools in the treatment of conditions associated with CNV, including neovascular AMD.

Pertussis toxin treatment counteracts intramembrane interactions between neuropeptide Y receptors and α2-adrenoceptors

The effect of intracerebroventricular injections of pertussis toxin were investigated on the neuropeptide Y-induced modulation of alpha 2-adrenoceptor binding in membranes from the dorsomedial medulla oblongata of the rat. Concentration-response experiments showed that neuropeptide Y reduced the binding affinity of the alpha 2-agonist, p-[3H]aminoclonidine, with a maximal effect of 30% at 3-30 nM. Pertussis toxin treatment (10 micrograms, 24 h) counteracted this modulation, without reducing the binding of neuropeptide Y to its own receptor. The results indicate that pertussis toxin-sensitive G-proteins are essential for the mediation of the intramembrane interaction between neuropeptide Y receptors and alpha 2-adrenoceptors.