Marlene A. Jacobson

Cardioprotection by Ecto-5'-Nucleotidase (CD73) and A2B Adenosine Receptors

Background— Ecto-5′-nucleotidase (CD73)–dependent adenosine generation has been implicated in tissue protection during acute injury. Once generated, adenosine can activate cell-surface adenosine receptors (A1AR, A2AAR, A2BAR, A3AR). In the present study, we define the contribution of adenosine to cardioprotection by ischemic preconditioning. Methods and Results— On the basis of observations of CD73 induction by ischemic preconditioning, we found that inhibition or targeted gene deletion of cd73 abolished infarct size-limiting effects. Moreover, 5′-nucleotidase treatment reconstituted cd73−/− mice and attenuated infarct sizes in wild-type mice. Transcriptional profiling of adenosine receptors suggested a contribution of A2BAR because it was selectively induced by ischemic preconditioning. Specifically, in situ ischemic preconditioning conferred cardioprotection in A1AR−/−, A2AAR−/−, or A3AR−/− mice but not in A2BAR−/− mice or in wild-type mice after inhibition of the A2BAR. Moreover, A2BAR agonist treatment significantly reduced infarct sizes after ischemia. Conclusions— Taken together, pharmacological and genetic evidence demonstrate the importance of CD73-dependent adenosine generation and signaling through A2BAR for cardioprotection by ischemic preconditioning and suggests 5′-nucleotidase or A2BAR agonists as therapy for myocardial ischemia.

N-desmethylclozapine, an allosteric agonist at muscarinic 1 receptor, potentiates N-methyl-d-aspartate receptor activity

The molecular and neuronal substrates conferring on clozapine its unique and superior efficacy in the treatment of schizophrenia remain elusive. The interaction of clozapine with many G protein-coupled receptors is well documented but less is known about its biologically active metabolite, N-desmethylclozapine. Recent clinical and preclinical evidences of the antipsychotic activity of the muscarinic agonist xanomeline prompted us to investigate the effects of N-desmethylclozapine on cloned human M1-M5 muscarinic receptors. N-desmethylclozapine preferentially bound to M1 muscarinic receptors with an IC50 of 55 nM and was a more potent partial agonist (EC50, 115 nM and 50% of acetylcholine response) at this receptor than clozapine. Furthermore, pharmacological and site-directed mutagenesis studies suggested that N-desmethylclozapine preferentially activated M1 receptors by interacting with a site that does not fully overlap with the acetylcholine orthosteric site. As hypofunction of N-methyl-d-aspartate (NMDA) receptor-driven neuronal ensembles has been implicated in psychotic disorders, the neuronal activity of N-desmethylclozapine was electrophysiologically investigated in hippocampal rat brain slices. N-desmethylclozapine was shown to dose-dependently potentiate NMDA receptor currents in CA1 pyramidal cells by 53% at 100 nM, an effect largely mediated by activation of muscarinic receptors. Altogether, our observations provide direct evidence that the brain penetrant metabolite N-desmethylclozapine is a potent, allosteric agonist at human M1 receptors and is able to potentiate hippocampal NMDA receptor currents through M1 receptor activation. These observations raise the possibility that N-desmethylclozapine contributes to clozapines clinical activity in schizophrenics through modulation of both muscarinic and glutamatergic neurotransmission.

Selective activation of the M1 muscarinic acetylcholine receptor achieved by allosteric potentiation

The forebrain cholinergic system promotes higher brain function in part by signaling through the M1 muscarinic acetylcholine receptor (mAChR). During Alzheimers disease (AD), these cholinergic neurons degenerate, therefore selectively activating M1 receptors could improve cognitive function in these patients while avoiding unwanted peripheral responses associated with non-selective muscarinic agonists. We describe here benzyl quinolone carboxylic acid (BQCA), a highly selective allosteric potentiator of the M1 mAChR. BQCA reduces the concentration of ACh required to activate M1 up to 129-fold with an inflection point value of 845 nM. No potentiation, agonism, or antagonism activity on other mAChRs is observed up to 100 μM. Furthermore studies in M1−/− mice demonstrates that BQCA requires M1 to promote inositol phosphate turnover in primary neurons and to increase c-fos and arc RNA expression and ERK phosphorylation in the brain. Radioligand-binding assays, molecular modeling, and site-directed mutagenesis experiments indicate that BQCA acts at an allosteric site involving residues Y179 and W400. BQCA reverses scopolamine-induced memory deficits in contextual fear conditioning, increases blood flow to the cerebral cortex, and increases wakefulness while reducing delta sleep. In contrast to M1 allosteric agonists, which do not improve memory in scopolamine-challenged mice in contextual fear conditioning, BQCA induces β-arrestin recruitment to M1, suggesting a role for this signal transduction mechanism in the cholinergic modulation of memory. In summary, BQCA exploits an allosteric potentiation mechanism to provide selectivity for the M1 receptor and represents a promising therapeutic strategy for cognitive disorders.

Adenosine and inosine increase cutaneous vasopermeability by activating A3 receptors on mast cells

Adenosine has potent effects on both the cardiovascular and immune systems. Exposure of tissues to adenosine results in increased vascular permeability and extravasation of serum proteins. The mechanism by which adenosine brings about these physiological changes is poorly defined. Using mice deficient in the A(3) adenosine receptor (A(3)AR), we show that increases in cutaneous vascular permeability observed after treatment with adenosine or its principal metabolite inosine are mediated through the A(3)AR. Adenosine fails to increase vascular permeability in mast cell-deficient mice, suggesting that this tissue response to adenosine is mast cell-dependent. Furthermore, this response is independent of activation of the high-affinity IgE receptor (FcepsilonR1) by antigen, as adenosine is equally effective in mediating these changes in FcepsilonR1 beta-chain-deficient mice. Together these results support a model in which adenosine and inosine induce changes in vascular permeability indirectly by activating mast cells, which in turn release vasoactive substances. The demonstration in vivo that adenosine, acting through a specific receptor, can provoke degranulation of this important tissue-based effector cell, independent of antigen activation of the high-affinity IgE receptor, supports an important role for this nucleoside in modifying the inflammatory response.

Activation of Murine Lung Mast Cells by the Adenosine A3 Receptor

Adenosine has been implicated to play a role in asthma in part through its ability to influence mediator release from mast cells. Most physiological roles of adenosine are mediated through adenosine receptors; however, the mechanisms by which adenosine influences mediator release from lung mast cells are not understood. We established primary murine lung mast cell cultures and used real-time RT-PCR and immunofluorescence to demonstrate that the A2A, A2B, and A3 adenosine receptors are expressed on murine lung mast cells. Studies using selective adenosine receptor agonists and antagonists suggested that activation of A3 receptors could induce mast cell histamine release in association with increases in intracellular Ca2+ that were mediated through Gi and phosphoinositide 3-kinase signaling pathways. The function of A3 receptors in vivo was tested by exposing mice to the A3 receptor agonist, IB-MECA. Nebulized IB-MECA directly induced lung mast cell degranulation in wild-type mice while having no effect in A3 receptor knockout mice. Furthermore, studies using adenosine deaminase knockout mice suggested that elevated endogenous adenosine induced lung mast cell degranulation by engaging A3 receptors. These results demonstrate that the A3 adenosine receptor plays an important role in adenosine-mediated murine lung mast cell degranulation.

Dose-dependent effect of CDPPB, the mGluR5 positive allosteric modulator, on recognition memory is associated with GluR1 and CREB phosphorylation in the prefrontal cortex and hippocampus.

In the search for strategies to treat schizophrenia, attention has focused on enhancing NMDA receptor function. In vitro experiments show that metabotropic glutamate 5 receptor (mGluR5) activation enhances NMDA receptor activity, and in vivo experiments indicate that mGluR5 positive allosteric modulators (PAMs) are effective in preclinical assays measuring antipsychotic potential and cognition. Here we characterized the dose-effect function of CDPPB (3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide), an mGluR5 PAM, on novel object recognition memory in unimpaired Wistar Hannover rats (0, 10 or 30 mg/kg CDPPB) and animals with an MK-801-induced deficit (0, 3, 10, or 30 mg/kg CDPPB). In each experiment compound was given 30 min prior to the first exposure in order to affect acquisition/consolidation of the memory. In both cases, an inverted-U-shaped dose-effect function was observed, with lower doses improving recognition but higher doses having no effect. We then examined the effects of CDPPB (0, 3, 10, or 30 mg/kg) on markers of synaptic plasticity in prefrontal cortex and hippocampus, focusing on the expression and phosphorylation status of proteins involved in NMDA related signaling, including the NMDA receptor subunits NR1 and NR2B, the AMPA receptor subunit GluR1, alphaCa((2+))/CaM dependent Ser-Thr kinases II (alphaCaMKII), and the transcription factor CREB. Expression and phosphorylation of many of these proteins, particularly in the prefrontal cortex, were also characterized by an inverted-U-shaped dose-effect function. Taken together, these findings show that mGluR5 activation enhances NMDA receptor function and markers of neuronal plasticity commensurate with improvements in recognition memory. However, the effects of CDPPB are heavily dependent on dose, with higher doses being ineffective in improving recognition memory and producing downstream effects consistent with heightened NMDA receptor activation. These findings may have important implications for the development of mGluR5 PAMs to treat schizophrenia.

A3 Adenosine Receptor Signaling Contributes to Airway Inflammation and Mucus Production in Adenosine Deaminase-Deficient Mice

Adenosine signaling has been implicated in chronic lung diseases such as asthma and chronic obstructive pulmonary disease; however, the specific roles of the various adenosine receptors in processes central to these disorders are not well understood. In this study, we have investigated the role(s) of the A3 adenosine receptor in adenosine-dependent pulmonary inflammation observed in adenosine deaminase (ADA)-deficient mice. The A3 receptor (A3R) was found to be expressed in eosinophils and mucus-producing cells in the airways of ADA-deficient mice. Treatment of ADA-deficient mice with MRS 1523, a selective A3R antagonist, prevented airway eosinophilia and mucus production. Similar findings were seen in the lungs of ADA/A3 double knockout mice. Although eosinophils were decreased in the airways of ADA-deficient mice following antagonism or removal of the A3R, elevations in circulating and lung interstitial eosinophils persisted, suggesting signaling through the A3R is needed for the migration of eosinophils into the airways. These findings identify an important role for the A3R in regulating lung eosinophilia and mucus production in an environment of elevated adenosine.

Adenosine A3 receptor stimulation inhibits migration of human eosinophils.

Activation of adenosine A3 receptors (A3‐R) produced a dose‐dependent reduction in the chemotaxis of human eosinophils to platelet‐activating factor (PAF), RANTES, and leukotriene B4 (LTB4) to a maximum of 58,48, and 52%, respectively (P < 0.02). This effect was completely reversed by selective A3‐R antagonists. In contrast, activation of A1 or A2a‐R did not affect PAF‐induced eosinophil chemotaxis. PAF upregulated the expression of CD11b/CD18, down‐regulated L‐selectin, and also increased F‐actin assembly in eosinophils. The expression of these activation markers was not influenced by A3‐R, A2a, or A1‐R stimulation. Activation of A3‐R may play an important role in inflammation by inhibiting eosinophil migration. J. Leukoc. Biol. 62: 465–468; 1997.

Cl-IB-MECA [2-Chloro-N6-(3-iodobenzyl)adenosine-5′-N-methylcarboxamide] Reduces Ischemia/Reperfusion Injury in Mice by Activating the A Adenosine Receptor

We used pharmacological agents and genetic methods to determine whether the potent A3 adenosine receptor (AR) agonist 2-chloro-N6-(3-iodobenzyl)adenosine-5′-N-methylcarboxamide (Cl-IB-MECA) protects against myocardial ischemia/reperfusion injury in mice via the A3AR or via interactions with other AR subtypes. Pretreating wild-type (WT) mice with Cl-IB-MECA reduced myocardial infarct size induced by 30 min of coronary occlusion and 24 h of reperfusion at doses (30 and 100 μg/kg) that concomitantly reduced blood pressure and stimulated systemic histamine release. The A3AR-selective antagonist MRS 1523 [3-propyl-6-ethyl-5[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridine-carboxylate], but not the A2AAR antagonist ZM 241385 [4-{2-7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl}phenol], blocked the reduction in infarct size provided by Cl-IB-MECA, suggesting a mechanism involving the A3AR. To further examine the selectivity of Cl-IB-MECA, we assessed its cardioprotective effectiveness in A3AR gene “knock-out” (A3KO) mice. Cl-IB-MECA did not reduce myocardial infarct size in A3KO mice in vivo and did not protect isolated perfused hearts obtained from A3KO mice from injury induced by global ischemia and reperfusion. Additional studies using WT mice treated with compound 48/80 [condensation product of p-methoxyphenethyl methylamine with formaldehyde] to deplete mast cell contents excluded the possibility that Cl-IB-MECA was cardioprotective by releasing mediators from mast cells. These data demonstrate that Cl-IB-MECA protects against myocardial ischemia/reperfusion injury in mice principally by activating the A3AR.

The Behavioral and Neurochemical Effects of a Novel d-Amino Acid Oxidase Inhibitor Compound 8 [4H-Thieno [3,2-b]pyrrole-5-carboxylic Acid] and d-Serine

Multiple studies indicate that N-methyl-d-aspartate (NMDA) receptor hypofunction underlies some of the deficits associated with schizophrenia. One approach for improving NMDA receptor function is to enhance occupancy of the glycine modulatory site on the NMDA receptor by increasing the availability of the endogenous coagonists d-serine. Here, we characterized a novel d-amino acid oxidase (DAAO) inhibitor, compound 8 [4H-thieno [3,2-b]pyrrole-5-carboxylic acid] and compared it with d-serine. Compound 8 is a moderately potent inhibitor of human (IC50, 145 nM) and rat (IC50, 114 nM) DAAO in vitro. In rats, compound 8 (200 mg/kg) decreased kidney DAAO activity by ∼96% and brain DAAO activity by ∼80%. This marked decrease in DAAO activity resulted in a significant (p < 0.001) elevation in both plasma (220% of control) and cerebrospinal fluid (CSF; 175% of control) d-serine concentration. However, compound 8 failed to significantly influence amphetamine-induced psychomotor activity, nucleus accumbens dopamine release, or an MK-801 (dizocilpine maleate)-induced deficit in novel object recognition in rats. In contrast, high doses of d-serine attenuated both amphetamine-induced psychomotor activity and dopamine release and also improved performance in novel object recognition. Behaviorally efficacious doses of d-serine (1280 mg/kg) increased CSF levels of d-serine 40-fold above that achieved by the maximal dose of compound 8. These findings demonstrate that pharmacological inhibition of DAAO significantly increases d-serine concentration in the periphery and central nervous system. However, acute inhibition of DAAO appears not to be sufficient to increase d-serine to concentrations required to produce antipsychotic and cognitive enhancing effects similar to those observed after administration of high doses of exogenous d-serine.