Eva Lindgren

Adenosine Receptors Mediating Cyclic AMP Productioin the Rat Hippocampus

Abstract: In the transversely cut rat hippocampus, adenosine caused a dose‐dependent increase in the accumulation of [3H]cyclic AMP from [3H]ATP. Adenosine breakdown products were inactive. AMP was somewhat less effective than adenosine, and its effect could be partially, but not completely, abolished by α,β‐methylene‐ADP and GMP, which inhibited its metabolism by 5′‐nucleotidase. The effect of adenosine was unaffected by inhibitors of adenosine deaminase, but enhanced by several inhibitors of adenosine uptake. Some analogues of adenosine, including N6‐phenylisopropyladenosine (PIA), 2‐chloroadenosine and adenosine 5′‐ethylcarboxamide (NECA), were more active than adenosine, whereas others such as 2‐deoxyadenosine and 9‐(tetrahydro‐2‐furyl)adenine (SQ 22536) actually inhibited the response. The effect of PIA was highly stereospecific. The action of adenosine was inhibited by several alkylxanthines, the most potent of which was 8‐phenyltheophylline. [3H]Cyclohexyladenosine (CHA) bound specifically to cell membranes from the rat hippocampus. The extent of binding was similar to that found in other cortical areas. The relative potency of some adenosine analogues and alkylxanthines to displace labelled CHA was essentially similar to their potency as effectors of the cyclic AMP system. Adenosine contributed to the cyclic AMP‐elevating effect of α‐adrenoceptor‐stimulating drugs and several amino acids, but not to that seen with isoprenaline. The cyclic AMP increase seen following depolarization was only partially adenosine‐dependent. The present results demonstrate that the rat hippocampus contains adenosine receptors mediating cyclic AMP accumulation and that these receptors have similar characteristics to those mediating pyramidal cell depression. Adenosine‐induced cyclic AMP accumulation may be used as a biochemical correlate to electrophysiology and as a convenient parameter to assess the influence of drugs on adenosine mechanisms in the rat hippocampus.

Effects of N‐ethylmaleimide and forskolin on glutamate release from rat hippocampal slices. Evidence that prejunctional adenosine receptors are linked to N‐proteins, but not to adenylate cyclase

In the present experiments we have examined the effect of N-ethylmaleimide (NEM) on the release of [3H]glutamate from rat hippocampal slices. Pretreatment of slices with NEM in a concentration between 50 microM and 200 microM, can inhibit the GTP-binding protein (Ni) that transmits receptor signals into inhibitions of adenylate cyclase, without affecting the Ns-protein, that transmits signals into stimulation of the cyclase, or the cyclase. The adenosine receptor agonist R-phenylisopropyladenosine (R-PIA, 1 microM) caused an approximately 50% inhibition of the evoked [3H]glutamate release. This effect was completely prevented by NEM treatment, which did not affect basal or stimulated release of the amino acid. By contrast, the effect of R-PIA was unaffected by adding an adenylate cyclase stimulator (forskolin 1 microM) and a phosphodiesterase inhibitor (rolipram, ZK 62.711, 30 microM) which raised the cyclic AMP content of the slices approximately 10-fold. In conclusion, these results suggest that the adenosine receptor that mediates prejunctional inhibition of glutamate release is coupled to a protein similar to the Ni-protein, but that another effector than adenylate cyclase is involved.

Adenosine A1 receptors regulate lipolysis and lipogenesis in mouse adipose tissue-interactions with insulin.

Adenosine acting at adenosine A1 receptors is considered to be one major regulator of adipose tissue physiology. We have examined the role of adenosine and its interactions with insulin in adipose tissue by using A1R knock out (-/-) mice. Removal of endogenous adenosine with adenosine deaminase caused lipolysis in A1R (+/+), but not A1R (-/-) adipocytes. The adenosine analogue, 2-chloroadenosine, inhibited noradrenaline-stimulated lipolysis and cAMP accumulation in A1R (+/+), but not in A1R (-/-) adipocytes. Insulin reduces lipolysis and cAMP via another mechanism than adenosine and acted additively, but not synergistically, with adenosine. Plasma levels of free fatty acids, glycerol and triglycerides were significantly lower in A1R (+/+) than in A1R (-/-) mice after administration of an adenosine analogue. 2-chloroadenosine induced lipogenesis in presence of insulin in A1R (+/+), but not in A1R (-/-) adipocytes. There were no changes in mRNA levels for several genes involved in fat synthesis in adipose tissue between genotypes. Body weight was similar in young A1R (+/+) and A1R (-/-) mice, but old male A1R (-/-) mice were heavier than wild type controls. In conclusion, adenosine inhibits lipolysis via the adenosine A1 receptor and other adenosine receptors play no significant role. Adenosine and insulin mediate additive but not synergistic antilipolytic effects and 2-chloroadenosine stimulates lipogenesis via adenosine A1 receptors. Thus deletion of adenosine A1 receptors should increase lipolysis and decrease lipogenesis, but in fact an increased fat mass was observed, indicating that other actions of adenosine A1 receptors, possibly outside adipose tissue, are also important.

Heterotrimeric G protein-dependent WNT-5A signaling to ERK1/2 mediates distinct aspects of microglia proinflammatory transformation

BackgroundWNT-5A signaling in the central nervous system is important for morphogenesis, neurogenesis and establishment of functional connectivity; the source of WNT-5A and its importance for cellular communication in the adult brain, however, are mainly unknown. We have previously investigated the inflammatory effects of WNT/β-catenin signaling in microglia in Alzheimers disease. WNT-5A, however, generally recruits β-catenin-independent signaling. Thus, we aim here to characterize the role of WNT-5A and downstream signaling pathways for the inflammatory transformation of the brains macrophages, the microglia.MethodsMouse brain sections were used for immunohistochemistry. Primary isolated microglia and astrocytes were employed to characterize the WNT-induced inflammatory transformation and underlying intracellular signaling pathways by immunoblotting, quantitative mRNA analysis, proliferation and invasion assays. Further, measurements of G protein activation by [γ-35 S]GTP binding, examination of calcium fluxes and cyclic AMP production were used to define intracellular signaling pathways.ResultsAstrocytes in the adult mouse brain express high levels of WNT-5A, which could serve as a novel astroglia-microglia communication pathway. The WNT-5A-induced proinflammatory microglia response is characterized by increased expression of inducible nitric oxide synthase, cyclooxygenase-2, cytokines, chemokines, enhanced invasive capacity and proliferation. Mapping of intracellular transduction pathways reveals that WNT-5A activates heterotrimeric Gi/o proteins to reduce cyclic AMP levels and to activate a Gi/o protein/phospholipase C/calcium-dependent protein kinase/extracellular signal-regulated kinase 1/2 (ERK1/2) axis. We show further that WNT-5A-induced ERK1/2 signaling is responsible for distinct aspects of the proinflammatory transformation, such as matrix metalloprotease 9/13 expression, invasion and proliferation.ConclusionsThus, WNT-5A-induced and G protein-dependent signaling to ERK1/2 is important for the regulation of proinflammatory responses in mouse primary microglia cells. We show for the first time that WNT-5A/G protein signaling mediates physiologically important processes in primary mammalian cells with natural receptor and G protein stochiometry. Consequently, WNT-5A emerges as an important means of astrocyte-microglia communication and we, therefore, suggest WNT-5A as a new player in neuroinflammatory conditions, such as neurodegenerative disease, hypoxia, stroke, injury and infection.

Treatment with N‐ethylmaleimide selectively reduces adenosine receptor‐mediated decreases in cyclic AMP accumulation in rat hippocampal slices

1 N‐ethylmaleimide (NEM) has been reported to interact with the GTP‐binding Ni‐protein; we have examined its effect on adenosine receptor binding in feline cortical membranes and on adenosine‐receptor mediated effects on cyclic AMP accumulation in rat hippocampal slices. 2 Treatment of cortical membranes with NEM (100 μM for 5 min) altered the binding of [3H]‐phenylisopropyladenosine (PIA) from being almost exclusively to a single class of high affinity sites (KD = 1.65 nM) to binding at two classes of sites (KDH = 2.1 nM, KDL = 102 nM). The total number of binding sites was similar (825–845 fmol mg−1 in control membranes, 944–1428 fmol mg−1 in NEM‐treated membranes). 3 In rat hippocampal slices treated with forskolin (0.3 μM) L‐PIA produced a biphasic effect on cyclic AMP accumulation: an inhibition at 0.03 to 1 μM and at higher concentrations, a stimulation. Treatment with 50 μM NEM selectively inhibited the inhibitory phase, causing stimulation at lower concentrations of L‐PIA. At 50 μM, NEM did not alter basal or forskolin‐stimulated cyclic AMP accumulation but at higher concentrations inhibition was observed. 4 It is concluded that NEM can, in certain doses, selectively block adenosine A1‐receptor‐mediated effects without affecting A2‐receptor‐mediated actions in the same tissue. It is suggested that this is due to NEM affecting the Ni guanine nucleotide binding protein.

Mice heterozygous for both A1 and A2A adenosine receptor genes show similarities to mice given long-term caffeine

Caffeine is believed to exert its stimulant effects by blocking A(2A) and A(1) adenosine receptors (A(2A)R and A(1)R). Although a genetic knockout of A(2A)R eliminates effects of caffeine, the phenotype of the knockout animal does not resemble that of caffeine treatment. In this study we explored the possibility that a mere reduction of the number of A(1)Rs and A(2A)Rs, achieved by deleting one of the two copies of the A(1)R and A(2A)R genes, would mimic some aspects of long-term caffeine ingestion. The A(1)R and A(2A)R double heterozygous (A(1)R-A(2A)R dHz) mice indeed had approximately one-half the number of A(1)R and A(2A)R, and there were little compensatory changes in A(2B) or A(3) adenosine receptor (A(2B)R or A(3)R) expression. The ability of a stable adenosine analog to activate receptors was shifted to the right by caffeine and in A(1)R-A(2A)R dHz tissue. Caffeine (0.3 g/l in drinking water for 7-10 days) and A(1)R-A(2A)R dHz genotype increased locomotor activity (LA) and decreased heart rate without significantly influencing body temperature. The acute stimulatory effect of a single injection of caffeine was reduced in A(1)R-A(2A)R dHz mice and in mice treated long term with oral caffeine. Thus at least some aspects of long-term caffeine use can be mimicked by genetic manipulation of the A(1)R and A(2A)R.

Eliminating the antilipolytic adenosine A1 receptor does not lead to compensatory changes in the antilipolytic actions of PGE2 and nicotinic acid

Aim:  We examined whether compensatory changes after adenosine A1 receptor knockout [A1R (−/−)] eliminate the antilipolytic actions mediated by this receptor.

Protein kinase C activation increases noradrenaline release from the rat hippocampus and modifies the inhibitory effect of α2-adrenoceptor and adenosine A1-receptor agonists

SummaryWe have studied the effect of stimulating protein kinase C with phorbol esters on the release of [3H]-noradrenaline (NA) in the absence or presence of presynaptic α2-adrenoceptor blocking agents and compared that to the elevation of cyclic AMP levels more than 10-fold by a combination of rolipram and forskolin. 4-β-Phorbol 12,13-dibutyrate (PDiBu) increased stimulated (3 Hz) [3H]-NA release markedly and in a concentration dependent manner. 4-α-Phorbol-12,13-didecanoate was ineffective. The effect of PDiBu was not significantly reduced by nifedipine (1 μM), but was proportionally less in the presence of an α2-adrenoceptor antagonist, yohimbine. PDiBu inhibited the presynaptic effect of α2-adrenoceptor agonists clonidine and UK 14304. By contrast, the presynaptic effect of the adenosine analogue R-PIA was not reduced by PDiBu. PDiBu caused an increase in cyclic AMP that depended on adenosine receptor stimulation. Elevation of cyclic AMP had a limited effect on NA release from rat hippocampus, and did not significantly decrease the presynaptic inhibitory effect of UK 14304 (0.1 μM), of morphine (1 μM) or of the adenosine A1-receptor agonist CHA (1 μM). The effect of phorbol esters and several presynaptic inhibitors of NA-release in the rat hippocampus cannot be explained by changes in cyclic AMP levels in the tissue. Phorbol esters that stimulate protein kinase C appear to interact with a target that is the site of action α2-adrenoceptors in this tissue. This site is not a dihydropyridine sensitive Ca-channel and is also different from the target of presynaptic adenosine receptors. Thus, activation of protein kinase C discriminates between apparently similar presynaptic mechanisms.

Interactions Between the Neuromodulator Adenosine and the Classic Transmitters

The present studies give examples of three types of interactions between adenosine and classic neurotransmitters. First, the transmitter substances may influence the amount of adenosine formed locally. Depolarizing stimuli have long been known to increase the amount of adenosine released from peripheral tissues as well as brain tissue in vitro and in vivo. In addition, the magnitude of this response, and the formation of adenosine induced by hypoxia, may be altered by other transmitters, such as opioids, in ways that are still undefined.

Formation and Actions of Adenosine in the Rat Hippocampus, with Special Reference to the Interactions with Classical Transmitters

Adenosine and related compounds can probably play a transmitter or cotransmitter role at least in some tissues (e.g., Burnstock, 1985). However, this seems to be rare and the major functional role of adenosine not only in the periphery but also in the nervous system appears to be that of a modulator. Adenosine is released not only from nerve terminals but also from cell bodies and from glial cells or endothelial cells lining the cerebral blood vessels. Besides its role in blood-flow regulation, adenosine clearly plays a role in modulation of nervous activity, pre- and postjunctionally.