Risperidone-induced inactivation and clozapine-induced reactivation of rat cortical astrocyte 5-hydroxytryptamine₇ receptors: evidence for in situ G protein-coupled receptor homodimer protomer cross-talk

We have reported previously novel drug-induced inactivation and reactivation of human 5-hydroxytryptamine? (5-HT?) receptors in a recombinant cell line. To explain these novel observations, a homodimer structure displaying protomer-protomer cross-talk was proposed. To determine whether these novel observations and interpretations are due to an artifactual G protein-coupled receptor (GPCR) mechanism unique to the recombinant cell line, we explored the properties of r5-HT? receptors expressed by cortical astrocytes in primary culture. As in the recombinant cell line, risperidone, 9-OH-risperidone, methiothepin, and bromocriptine were found to potently inactivate r5-HT? receptors. As in the recombinant cell line, exposure of risperidone-inactivated astrocyte r5-HT? receptors to competitive antagonists resulted in the reactivation of r5-HT? receptors. The potencies of the reactivating drugs closely correlated with their affinities for h5-HT? receptors. These results indicate the novel inactivating and reactivating property of drugs is not due to an artifact of the recombinant cell line expressing h5-HT? receptors but is an intrinsic property of 5-HT? receptors in vitro and ex vivo. This evidence suggests that a native (nonmutated) GPCR, in its native membrane environment (cortical astrocyte primary culture), can function as a homodimer with protomer-protomer cross-talk. Homodimers may be a common GPCR structure. The experimental design used in our studies can be used to explore the properties of other GPCRs in their native forms in recombinant cells, primary cultures expressing the endogenous GPCRs, and possibly in vivo. The homodimer structure and protomer-protomer cross-talk offer new avenues of research into receptor dysfunction in disease states and the development of novel drugs.     

Design and characterization of optimized adenosine A₂A/A₁ receptor antagonists for the treatment of Parkinson's disease

The design and characterization of two, dual adenosine A(2A)/A(1) receptor antagonists in several animal models of Parkinson's disease is described. Compound 1 was previously reported as a potential treatment for Parkinson's disease. Further characterization of 1 revealed that it was metabolized to reactive intermediates that caused the genotoxicity of 1 in the Ames and mouse lymphoma L51784 assays. The identification of the metabolites enabled the preparation of two optimized compounds 13 and 14 that were devoid of the metabolic liabilities associated with 1. Compounds 13 and 14 are potent dual A(2A)/A(1) receptor antagonists that have excellent activity, after oral administration, across a number of animal models of Parkinson's disease including mouse and rat models of haloperidol-induced catalepsy, mouse and rat models of reserpine-induced akinesia, and the rat 6-hydroxydopamine (6-OHDA) lesion model of drug-induced rotation.     

Agonist activation of the G protein-coupled receptor GPR35 involves transmembrane domain III and is transduced via Gα₁₃ and β-arrestin-2

BACKGROUND AND PURPOSE

GPR35 is a poorly characterized G protein-coupled receptor at which kynurenic acid has been suggested to be the endogenous ligand. We wished to test this and develop assays appropriate for the study of this receptor.

EXPERIMENTAL APPROACH

Human and rat orthologues of GPR35 were engineered and expressed and assays developed to assess interaction with β-arrestin-2, activation of Gα₁₃ and agonist-induced internalization.

KEY RESULTS

GPR35-β-arrestin-2 interaction assays confirmed that both the endogenous tryptophan metabolite kynurenic acid and the synthetic ligand zaprinast had agonist action at each orthologue. Zaprinast was substantially more potent than kynurenic acid at each and both agonists displayed substantially greater potency at rat GPR35. Two novel thiazolidinediones also displayed agonism and displayed similar potency at each GPR35 orthologue. The three ligand classes acted orthosterically with respect to each other, suggesting overlapping binding sites and, consistent with this, mutation to alanine of the conserved arginine at position 3.36 or tyrosine 3.32 in transmembrane domain III abolished β-arrestin-2 recruitment in response to each ligand at each orthologue.

CONCLUSIONS AND IMPLICATIONS

These studies indicate that β-arrestin-2 interaction assays are highly appropriate to explore the pharmacology of GPR35 and that Gα₁₃ activation is an alternative avenue of signal generation from GPR35. Arginine and tyrosine residues in transmembrane domain III are integral to agonist recognition and function of this receptor. The potency of kynurenic acid at human GPR35 is sufficiently low, however, to question whether it is likely to be the true endogenous ligand for this receptor.     

Does adenosine A1 receptor stimulation causes QRS prolongation by blocking beta adrenergic receptors in amitriptyline poisoning?

Aims

To investigate the role of beta receptor blockade via adenosine A₁ receptor stimulation on amitriptyline-induced QRS prolongation.

Methods

Isolated rat hearts were randomized into three groups (n = 8 for each group). After pretreatment with 5% dextrose (control) or DPCPX (8-cyclopentyl-1,3-dipropylxanthine), or propranolol + DPCPX, amitriptyline infusion was given to all groups. Intact beta adrenergic receptor response was verified with a bolus dose of isoproteranol (3 × 10⁻⁵ M).

Results

Amitriptyline (5.5 × 10⁻⁵ M) infusion following pretreatment with 5% dextrose or 10⁻⁴ M DPCPX prolonged QRS by 40–110% and 30–75%, respectively. After the beta receptor blockade with 10⁻² M propranolol bolus, amitriptyline infusion following pretreatment with DPCPX prolonged QRS by 40–130%. Amitriptyline infusion following pretreatment with DPCPX (10⁻⁴ M) shortened the QRS at 40, 50 and 60  min significantly when compared to propranolol + DPCPX group (168.8 ± 4.9%, p < 0.05; 170.8 ± 6.9%, p < 0.01; 174.0 ± 6.9%, p < 0.01, respectively). Amitriptyline infusion following pretreatment with 5% dextrose prolonged QRS duration significantly at 50th minutes (209.5 ± 6.1%, p < 0.05) compared to DPCPX pretreatment group.

Conclusion

DPCPX pretreatment shortened amitriptyline-induced QRS prolongation. Beta adrenergic receptor blockade enhanced QRS prolongation shortened by DPCPX pretreatment. Adenosine A₁ receptor stimulation related to beta adrenergic receptor blockade may play a role in amitriptyline-induced QRS prolongation in isolated rat hearts.     

Functional interaction between pre-synaptic α6β2-containing nicotinic and adenosine A2A receptors in the control of dopamine release in the rat striatum

Background and Purpose

Pre-synaptic nicotinic ACh receptors (nAChRs) and adenosine A_2A receptors (A_2ARs) are involved in the control of dopamine release and are putative therapeutic targets in Parkinson''s disease and addiction. Since A_2ARs have been reported to interact with nAChRs, here we aimed at mapping the possible functional interaction between A_2ARs and nAChRs in rat striatal dopaminergic terminals.

Experimental Approach

We pharmacologically characterized the release of dopamine and defined the localization of nAChR subunits in rat striatal nerve terminals in vitro and carried out locomotor behavioural sensitization in rats in vivo.

Key Results

In striatal nerve terminals, the selective A_2AR agonist {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 inhibited, while the A_2AR antagonist ZM241385 potentiated the nicotine-stimulated [³H]dopamine ([³H]DA) release. Upon blockade of the α6 subunit-containing nAChRs, the remaining nicotine-stimulated [³H]DA release was no longer modulated by A_2AR ligands. In the locomotor sensitization experiments, nicotine enhanced the locomotor activity on day 7 of repeated nicotine injection, an effect that no longer persisted after 1 week of drug withdrawal. Notably, ZM241385-injected rats developed locomotor sensitization to nicotine already on day 2, which remained persistent upon nicotine withdrawal.

Conclusions and Implications

These results provide the first evidence for a functional interaction between nicotinic and adenosine A_2AR in striatal dopaminergic terminals, with likely therapeutic consequences for smoking, Parkinson''s disease and other dopaminergic disorders.     

Why are A(2B) receptors low-affinity adenosine receptors? Mutation of Asn273 to Tyr increases affinity of human A(2B) receptor for 2-(1-Hexynyl)adenosine

Adenosine A(2B) receptors are known as low-affinity receptors due to their modest-to-negligible affinity for adenosine and prototypic agonists. Despite numerous synthetic efforts, 5'-N-ethylcarboxamidoadenosine (NECA) still is the reference agonist, albeit nonselective for this receptor. In our search for higher affinity agonists, we developed decision schemes to select amino acids for mutation to the corresponding residues in the most homologous, higher affinity, human A(2A) receptor. One scheme exploited knowledge on sequence alignments and modeling data and yielded three residues, V11, L58, and F59, mutation of which did not affect agonist affinity. The second scheme combined knowledge on sequence alignments and mutation data and pointed to Ala12 and Asn273. Mutation of Ala12 to threonine did not affect the affinity for NECA, (R)-N(6)-(phenylisopropyl)adenosine (R-PIA), and 2Cl Ado. The affinity of the N273Y mutant for NECA and R-PIA and for the antagonists xanthine amine congener (XAC), ZM241385, and SCH58261 was also unaltered. However, this mutant had a slightly increased affinity for a 2-substituted adenosine derivative, CGS21680. This prompted us to investigate other 2-substituted adenosines, with selectivity and high affinity for A(2A) receptors. All four compounds tested had improved affinity for the N273Y receptor. Of these, 2-(1-hexynyl)adenosine had submicromolar affinity for the N273Y receptor, 0.18 +/- 0.10 microM, with a 61-fold affinity gain over the wt receptor. In addition, the non-NECA analog (S)-PHP adenosine had an affinity of 1.7 +/- 0.5 microM for the wt receptor. The high affinity of (S)-PHP adenosine for the wt receptor suggests that further modifications at the 2-position may yield agonists with even higher affinity for A(2B) receptors.     

Spermidine decreases Na⁺,K⁺-ATPase activity through NMDA receptor and protein kinase G activation in the hippocampus of rats

Spermidine is an endogenous polyamine with a polycationic structure present in the central nervous system of mammals. Spermidine regulates biological processes, such as Ca(2+) influx by glutamatergic N-methyl-d-aspartate receptor (NMDA receptor), which has been associated with nitric oxide synthase (NOS) and cGMP/PKG pathway activation and a decrease of Na(+),K(+)-ATPase activity in rats' cerebral cortex synaptosomes. Na(+),K(+)-ATPase establishes Na(+) and K(+) gradients across membranes of excitable cells and by this means maintains membrane potential and controls intracellular pH and volume. However, it has not been defined whether spermidine modulates Na(+),K(+)-ATPase activity in the hippocampus. In this study we investigated whether spermidine alters Na(+),K(+)-ATPase activity in slices of hippocampus from rats, and possible underlying mechanisms. Hippocampal slices and homogenates were incubated with spermidine (0.05-10 μM) for 30 min. Spermidine (0.5 and 1 μM) decreased Na(+),K(+)-ATPase activity in slices, but not in homogenates. MK-801 (100 and 10 μM), a non-competitive antagonist of NMDA receptor, arcaine (0.5μM), an antagonist of the polyamine binding site at the NMDA receptor, and L-NAME (100μM), a NOS inhibitor, prevented the inhibitory effect of spermidine (0.5 μM). ODQ (10 μM), a guanylate cyclase inhibitor, and KT5823 (2 μM), a protein kinase G inhibitor, also prevented the inhibitory effect of spermidine on Na(+),K(+)-ATPase activity. Spermidine (0.5 and 1.0 μM) increased NO(2) plus NO(3) (NOx) levels in slices, and MK-801 (100 μM) and arcaine (0.5 μM) prevented the effect of spermidine (0.5 μM) on the NOx content. These results suggest that spermidine-induced decrease of Na(+),K(+)-ATPase activity involves NMDA receptor/NOS/cGMP/PKG pathway.     

Transmembrane α-helix 2 and 7 are important for small molecule-mediated activation of the GLP-1 receptor

Glucagon-like peptide-1 (GLP-1) activates the GLP-1 receptor (GLP-1R), which belongs to family B of the G-protein-coupled receptors. We previously identified a selective small molecule ligand, compound 2, that acted as a full agonist and allosteric modulator of GLP-1R. In this study, the structurally related small molecule, compound 3, stimulated cAMP production from GLP-1R, but not from the homologous glucagon receptor (GluR). The receptor selectivity encouraged a chimeric receptor approach to identify domains important for compound 3-mediated activation of GLP-1R. A subsegment of the GLP-1R transmembrane domain containing TM2 to TM5 was sufficient to transfer compound 3 responsiveness to GluR. Therefore, divergent residues in this subsegment of GLP-1R and GluR are responsible for the receptor selectivity of compound 3. Functional analyses of other chimeric receptors suggested that the existence of a helix-helix interface between TM1 and TM7 is important for the compound 3 response. Furthermore, site-directed mutagenesis revealed that a Phe195-Leu substitution in TM2 and a Thr391-Ala substitution in TM7 increased and decreased the efficacy of compound 3 without disturbing the potency or efficacy of GLP-1. Collectively, differential effects of receptor mutations suggest that TM2 and/or TM7 are important for compound 3-mediated activation of GLP-1R.     

Role of the A2B receptor–adenosine deaminase complex in colonic dysmotility associated with bowel inflammation in rats

BACKGROUND AND PURPOSE

Adenosine A_2B receptors regulate several physiological enteric functions. However, their role in the pathophysiology of intestinal dysmotility associated with inflammation has not been elucidated. Hence, we investigated the expression of A_2B receptors in rat colon and their role in the control of cholinergic motility in the presence of bowel inflammation.

EXPERIMENTAL APPROACH

Colitis was induced by 2,4-dinitrobenzenesulfonic acid (DNBS). Colonic A_2B receptor expression and localization were examined by RT-PCR and immunofluorescence. The interaction between A_2B receptors and adenosine deaminase was assayed by immunoprecipitation. The role of A_2B receptors in the control of colonic motility was examined in functional experiments on longitudinal muscle preparations (LMPs).

KEY RESULTS

A_2B receptor mRNA was present in colon from both normal and DNBS-treated rats but levels were increased in the latter. A_2B receptors were predominantly located in the neuromuscular layer, but, in the presence of colitis, were increased mainly in longitudinal muscle. Functionally, the A_2B receptor antagonist MRS 1754 enhanced both electrically-evoked and carbachol-induced cholinergic contractions in normal LMPs, but was less effective in inflamed tissues. The A_2B receptor agonist NECA decreased colonic cholinergic motility, with increased efficacy in inflamed LMP. Immunoprecipitation and functional tests revealed a link between A_2B receptors and adenosine deaminase, which colocalize in the neuromuscular compartment.

CONCLUSIONS AND IMPLICATIONS

Under normal conditions, endogenous adenosine modulates colonic motility via A_2B receptors located in the neuromuscular compartment. In the presence of colitis, this inhibitory control is impaired due to a link between A_2B receptors and adenosine deaminase, which catabolizes adenosine, thus preventing A_2B receptor activation.     

Role for engagement of β‐arrestin2 by the transactivated EGFR in agonist‐specific regulation of δ receptor activation of ERK1/2

Background and Purpose

β‐Arrestins function as signal transducers linking GPCRs to ERK1/2 signalling either by scaffolding members of ERK1/2s cascades or by transactivating receptor tyrosine kinases through Src‐mediated release of transactivating factor. Recruitment of β‐arrestins to the activated GPCRs is required for ERK1/2 activation. Our previous studies showed that δ receptors activate ERK1/2 through a β‐arrestin‐dependent mechanism without inducing β‐arrestin binding to the δ receptors. However, the precise mechanisms involved remain to be established.

Experimental Approach

ERK1/2 activation by δ receptor ligands was assessed using HEK293 cells in vitro and male Sprague Dawley rats in vivo. Immunoprecipitation, immunoblotting, siRNA transfection, intracerebroventricular injection and immunohistochemistry were used to elucidate the underlying mechanism.

Key Results

We identified a new signalling pathway in which recruitment of β‐arrestin2 to the EGFR rather than δ receptor was required for its role in δ receptor‐mediated ERK1/2 activation in response to H‐Tyr–Tic–Phe–Phe–OH (TIPP) or morphine stimulation. Stimulation of the δ receptor with ligands leads to the phosphorylation of PKCδ, which acts upstream of EGFR transactivation and is needed for the release of the EGFR‐activating factor, whereas β‐arrestin2 was found to act downstream of the EGFR transactivation. Moreover, we demonstrated that coupling of the PKCδ/EGFR/β‐arrestin2 transactivation pathway to δ receptor‐mediated ERK1/2 activation was ligand‐specific and the Ser³⁶³ of δ receptors was crucial for ligand‐specific implementation of this ERK1/2 activation pathway.

Conclusions and Implications

The δ receptor‐mediated activation of ERK1/2 is via ligand‐specific transactivation of EGFR. This study adds new insights into the mechanism by which δ receptors activate ERK1/2.

Abbreviations

DPDPE

[D‐Pen2, D‐Pen5] enkephalin

HB‐EGF

heparin‐binding EGF‐like growth factor

IGFR

insulin‐like growth factor receptor

NG108‐15

cell mouse neuroblastoma x rat glioma hybrid cell

RTK

receptor tyrosine kinase

TIPP

H‐Tyr‐Tic‐Phe‐Phe‐OH

     

A search for presynaptic imidazoline receptors at rabbit and rat noradrenergic neurones in the absence of α2-autoinhibition

Presynaptic, release-inhibiting imidazoline receptors have hitherto been detected mainly under conditions of ongoing α₂-autoinhibition. We tried to find them under α₂-autoinhibition-free conditions, in the majority of experiments in the rabbit pulmonary artery but in a few experiments also in rabbit atria, rabbit brain cortex and rat brain cortex. Tissue segments were preincubated with [³H]noradrenaline and then superfused and stimulated electrically. Ten compounds, some thought to inhibit noradrenaline release through activation of presynaptic imidazoline receptors, some thought to act through α₂-adrenoceptors, were tested. Rauwolscine and 6-chloro-N-methyl-2,3,4,5-tetrahydro-1-H-3-benzazepine (SKF 86466) were used as antagonists. In rabbit pulmonary artery segments stimulated by trains of 20 pulses/50 Hz (α₂-autoinhibition-free conditions), all ten “agonists” [medetomidine, aganodine, 4-chloro-2-guanidyl-isoindoline (BDF 7579), 4-chloro-2-(2-imidazolin-2-ylamino)-isoindoline (BDF 6143), 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14304), α-methylnoradrenaline, clonidine, moxonidine, cirazoline and idazoxan] reduced the stimulation-evoked overflow of tritium, with potency decreasing in that order and with maximal inhibition by 59% (idazoxan) to 95% (moxonidine). Rauwolscine competitively antagonized the effects of all ten “agonists” with similar potency, the pK _d-values lying between 7.6 and 8.2. Similarly, SKF 86466 competitively antagonized the effects of clonidine and moxonidine with the same potency (pK _d 7.6). Cirazoline was also studied in the other three tissues. In rabbit atrial segments stimulated by 20 pulses/50 Hz and rabbit brain cortex slices stimulated by 4 pulses/100 Hz (both autoinhibition-free), cirazoline reduced the evoked overflow of tritium with concentration-inhibition curves similar to the curve in the pulmonary artery. In the brain cortex, the pK _d-value of rauwolscine against cirazoline was 7.7 (pulmonary artery: 7.6). In rat brain cortex slices stimulated by 3 pulses/100 Hz (autoinhibition-free), cirazoline failed to change the evoked overflow of tritium but antagonized the inhibitory effect of UK 14304, pK _d of cirazoline against UK 14304 6.9. In rat brain cortex slices stimulated by trains of 36 pulses/3 Hz, finally (marked α₂-autoinhibition), cirazoline increased the evoked overflow of tritium. In the rabbit pulmonary artery, rauwolscine and SKF 86466 acted with the same potency against typical presynaptic imidazoline receptor agonists (such as clonidine) and typical α₂-adrenoceptor agonists (such as moxonidine). Hence, presynaptic imidazoline receptors were not detectable, in a tissue that is prototypical for presynaptic imidazoline receptors, in the absence of α₂-autoinhibition, i.e. under conditions usually thought to be optimal for presynaptic receptor characterization. The pK _d-values of rauwolscine and SKF 86466 indicate that all ten agonists activated the α_2A-autoreceptors of the pulmonary artery. Cirazoline behaved as an α_2(A)-autoreceptor agonist in rabbit tissues but as an α_2(D)-autoreceptor antagonist in rat tissues. Perhaps cirazoline generally possesses greater intrinsic activity at (human, rabbit) α_2A-adrenoceptors than the orthologous (rodent) α_2D-adrenoceptors. Received: 15 July 1998 / Accepted: 19 November 1998     

Structure–activity relationships of the N-terminus of calcitonin gene-related peptide: key roles of alanine-5 and threonine-6 in receptor activation

Background and Purpose: The N-terminus of calcitonin gene-related peptide (CGRP) is important for receptor activation, especially the disulphide-bonded ring (residues 1–7). However, the roles of individual amino acids within this region have not been examined and so the molecular determinants of agonism are unknown. This study has examined the role of residues 1, 3–6 and 8–9, excluding Cys-2 and Cys-7.Experimental Approach: CGRP derivatives were substituted with either cysteine or alanine; further residues were introduced at position 6. Their affinity was measured by radioligand binding and their efficacy by measuring cAMP production in SK-N-MC cells and β-arrestin 2 translocation in CHO-K1 cells at the CGRP receptor.Key Results: Substitution of Ala-5 by cysteine reduced affinity 270-fold and reduced efficacy for production of cAMP in SK-N-MCs. Potency at β-arrestin translocation was reduced by ninefold. Substitution of Thr-6 by cysteine destroyed all measurable efficacy of both cAMP and β-arrestin responses; substitution with either alanine or serine impaired potency. Substitutions at positions 1, 4, 8 and 9 resulted in approximately 10-fold reductions in potency at both responses. Similar observations were made at a second CGRP-activated receptor, the AMY_1(a) receptor.Conclusions and Implications: Ala-5 and Thr-6 are key determinants of agonist activity for CGRP. Ala-5 is also very important for receptor binding. Residues outside of the 1–7 ring also contribute to agonist activity.     

The dopamine receptor D2 agonist bromocriptine inhibits glucose-stimulated insulin secretion by direct activation of the α2-adrenergic receptors in beta cells

Treatment with the dopamine receptor D2 (DRD2) agonist bromocriptine improves metabolic features in obese patients with type 2 diabetes by a still unknown mechanism. In the present study, we investigated the acute effect of bromocriptine and its underlying mechanism(s) on insulin secretion both in vivo and in vitro. For this purpose, C57Bl6/J mice were subjected to an intraperitoneal glucose tolerance test (ipGTT) and a hyperglycemic (HG) clamp 60 min after a single injection of bromocriptine or placebo. The effects of bromocriptine on glucose-stimulated insulin secretion (GSIS), cell membrane potential and intracellular cAMP levels were also determined in INS-1E beta cells. We report here that bromocriptine increased glucose levels during ipGTT in vivo, an effect associated with a dose-dependent decrease in GSIS. During the HG clamp, bromocriptine reduced both first-phase and second-phase insulin response. This inhibitory effect was also observed in INS-1E beta cells, in which therapeutic concentrations of bromocriptine (0.5-50 nM) decreased GSIS. Mechanistically, neither cellular energy state nor cell membrane depolarization was affected by bromocriptine whereas intracellular cAMP levels were significantly reduced, suggesting involvement of G-protein-coupled receptors. Surprisingly, the DRD2 antagonist domperidone did not counteract the effect of bromocriptine on GSIS, whereas yohimbine, an antagonist of the α2-adrenergic receptors, completely abolished bromocriptine-induced inhibition of GSIS. In conclusion, acute administration of bromocriptine inhibits GSIS by a DRD2-independent mechanism involving direct activation of the pancreatic α2-adrenergic receptors. We suggest that treatment with bromocriptine promotes beta cells rest, thereby preventing long-lasting hypersecretion of insulin and subsequent beta cell failure.     

Involvement of central β2-adrenergic, NMDA and thromboxane A2 receptors in the pressor effect of anandamide in rats

Intravenous (i.v.) injection of the endocannabinoid anandamide induces triphasic cardiovascular responses, including a pressor effect mediated via unknown central and peripheral mechanism(s). The aim of the present study was to determine the central mechanism(s) responsible for the pressor response to anandamide. For this purpose, the influence of antagonists at thromboxane A₂ TP (sulotroban, daltroban, SQ 29548), NMDA (MK-801) and β₂-adrenergic receptors (ICI 118551) on the pressor effect induced by i.v. and intracerebroventricularly (i.c.v.) administered anandamide was examined in urethane-anaesthetized rats. Anandamide (1.5–3 µmol/kg, i.v.) or its stable analogue methanandamide (0.75 µmol/kg, i.v.) increased blood pressure by 25%. Anandamide (0.03 μmol per animal i.c.v.) caused a pure pressor effect (by 20%) but only in the presence of antagonists of CB₁ and TRPV1 receptors. The effects of cannabinoids (i.v. or i.c.v.) were diminished by i.v. daltroban, sulotroban (10 μmol/kg each), and/or SQ 29548 (1 μmol/kg). The effect of anandamide i.v. was reduced by SQ 29548 (0.02 μmol per animal i.c.v.) and by the thromboxane A₂ synthesis inhibitor furegrelate i.c.v. (1.8 µmol per animal). ICI 118551, MK-801 (1 µmol/kg i.v. each), and bilateral adrenalectomy diminished the effect of anandamide i.c.v. Sulotroban (i.v.) failed to affect the response to anandamide (i.v.) in pithed rats, and anandamide and methanandamide did not bind to TP receptors in rat platelets. The present study suggests that central β₂-adrenergic, NMDA and thromboxane A₂ receptors are involved in the anandamide-induced adrenal secretion of catecholamines and their pressor effect in urethane-anaesthetized rats.     

Activation of adenosine A2A receptor reduces osteoclast formation via PKA- and ERK1/2-mediated suppression of NFκB nuclear translocation

Background and Purpose

We previously reported that adenosine, acting at adenosine A_2A receptors (A_2AR), inhibits osteoclast (OC) differentiation in vitro (A_2AR activation OC formation reduces by half) and in vivo. For a better understanding how adenosine A_2AR stimulation regulates OC differentiation, we dissected the signalling pathways involved in A_2AR signalling.

Experimental Approach

OC differentiation was studied as TRAP+ multinucleated cells following M-CSF/RANKL stimulation of either primary murine bone marrow cells or the murine macrophage line, RAW264.7, in presence/absence of the A_2AR agonist {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680, the A_2AR antagonist ZM241385, PKA activators (8-Cl-cAMP 100 nM, 6-Bnz-cAMP) and the PKA inhibitor (PKI). cAMP was quantitated by EIA and PKA activity assays were carried out. Signalling events were studied in PKA knockdown (lentiviral shRNA for PKA) RAW264.7 cells (scrambled shRNA as control). OC marker expression was studied by RT-PCR.

Key Results

A_2AR stimulation increased cAMP and PKA activity which and were reversed by addition of ZM241385. The direct PKA stimuli 8-Cl-cAMP and 6-Bnz-cAMP inhibited OC maturation whereas PKI increased OC differentiation. A_2AR stimulation inhibited p50/p105 NFκB nuclear translocation in control but not in PKA KO cells. A_2AR stimulation activated ERK1/2 by a PKA-dependent mechanism, an effect reversed by ZM241385, but not p38 and JNK activation. A_2AR stimulation inhibited OC expression of differentiation markers by a PKA-mechanism.

Conclusions and Implications

A_2AR activation inhibits OC differentiation and regulates bone turnover via PKA-dependent inhibition of NFκB nuclear translocation, suggesting a mechanism by which adenosine could target bone destruction in inflammatory diseases like Rheumatoid Arthritis.