Hippocampal Serotonin 5-HT1A Receptor Enhances Acetylcholine Release in Conscious Rats

Abstract: We investigated changes in the extracellular levels of acetylcholine (ACh) following local application of serotonergic agents to the dorsal hippocampus of freely moving rats by means of perfusion using a microdialysis technique. Perfusion of serotonin (5-HT; 10 μM, for 30 min at a rate of 3 μl/min), dissolved in Ringer's solution containing 10 μM eserine, showed no marked effect on the extracellular levels of ACh. 8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; 20 μM), a 5-HT_1A agonist, increased ACh levels, whereas 7-trifluoromethyl-4-(4-methyl-1 -piperazinyl)-pymoto[1,2-a]quinoxaline (CGS-12066B; 100 μM), a 5-HT_1B agonist, decreased it. Clomipramine (2 μM), an uptake inhibitor of 5-HT, had no effect on ACh levels. Following perfusion of 1-(2-methoxyphenyl)-4-[4- (2-phthalimido)butyl]piperazine (NAN-190; 10 μM), which is a selective 5-HT_1A antagonist, the effect of 8-OH-DPAT was totally abolished, whereas CGS-12066B decreased extracellular ACh levels. 5-HT, as well as Clomipramine, had a decreasing effect on ACh levels after pretreatment with NAN-190. These results indicate that the 5-HT_1A receptor, which exists in the dorsal hippocampus, enhances the spontaneous ACh release, and that the mechanism of serotonergic modulation of ACh release partly depends on both the stimulatory control via the 5-HT_1A receptor and the suppressive one via the 5-HT_1B receptor in the dorsal hippocampus of rats.     

The Adenosine Receptor Agonist,APNEA, Increases Calcium Influx into Rat Cortical Synaptosomes through N-Type Channels Associated with A2a Receptors

N⁶-2-(4-aminophenyl)ethyladenosine (APNEA) is a nonselective adenosine receptor agonist known to have a high affinity for the adenosine A₁ and A₃ receptors. It was found to be able to dose-dependently increase the sustained (4 min) Ca²⁺ influx into rat cortical synaptosomes while 2-chloro-N⁶-(3-iodobenzyl)-adenosine-5-N-methyluronamide (Cl-IB-MECA), a selective A₃ agonist has no effect. However, this effect of APNEA was not affected by the presence of 8-cyclopentyl-l,3-dimethylxanthine (CPT), a selective A₁ antagonist; but instead completely abolished by 8-(3-chlorostyryl)caffeine (CSC), a selective A_2a antagonist, or -conotoxin GVIA. These results show that in the rat cortex, presynaptic A_2a receptors can mediate neurotransmitter release by increasing Ca²⁺ influx through the N-type calcium channels. A₁ and A₃ receptors appear not to be involved.     

Excitatory and Inhibitory Effects of A1 and A2A Adenosine Receptor Activation on the Electrically Evoked [3H]Acetylcholine Release from Different Areas of the Rat Hippocampus

Abstract: The modulation by adenosine analogues and endogenous adenosine of the electrically evoked release of [³H]acetylcholine ([³H]ACh) was compared in subslices of the three areas of the rat hippocampus (CA1, CA3, and dentate gyrus). The mixed A₁/A₂ agonist 2-chloroadenosine (CADO; 2–10 µM) inhibited, in a concentration-dependent manner, the release of [³H]ACh from the three hippocampal areas, being more potent in the CA1 and CA3 areas than in the dentate gyrus. The inhibitory effect of CADO (5 µM) on [³H]ACh release was prevented by the A₁ antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 50 nM) in the three hippocampal areas and was converted in an excitatory effect in the CA3 and dentate gyrus areas. The A_2A agonist CGS-21680 (30 nM) produced a greater increase of the evoked release of [³H]ACh in the CA3 than in the dentate gyrus areas, whereas no consistent effect was found in the CA1 area or in the whole hippocampal slice. The excitatory effect of CGS-21680 (30 nM) in the CA3 area was prevented by the adenosine receptor antagonist 3,7-dimethyl-1-propargylxanthine (10 µM). Both adenosine deaminase (2 U/ml) and DPCPX (250 nM) increased the evoked release of [³H]ACh in the CA1 and CA3 areas but not in the dentate gyrus. The amplitude of the effect of DPCPX and adenosine deaminase was similar in the CA1 area, but in the CA3 area DPCPX produced a greater effect than adenosine deaminase. It is concluded that the electrically evoked release of [³H]ACh in the three areas of the rat hippocampus can be differentially modulated by adenosine. In the CA1 area, only A₁ inhibitory receptors modulate ACh release, whereas in the CA3 area, both A_2A excitatory and A₁ inhibitory adenosine receptors modulate ACh release. In the dentate gyrus, both A₁ inhibitory and A_2A excitatory adenosine receptors are present, but endogenous adenosine does not activate them.     

Adenosine A(1) receptors mediate plasma exudation from the oral mucosa.

The purpose of this study was to pharmacologically characterize the adenosine receptor subtype(s) that mediates adenosine-induced increases in macromolecular efflux from the intact hamster cheek pouch. Using intravital microscopy, we found that 1,3-dipropyl-8-(2-amino-4-chlorophenyl)-xanthine (PACPX), a selective adenosine receptor-1 antagonist, but not 3,7-dimethyl-1-propargylxanthine (DMPX), a selective adenosine receptor-2 antagonist, significantly attenuated adenosine-induced leaky site formation and increased clearance of fluorescein isothiocyanate-labeled dextran (molecular mass, 70 kDa) from the intact hamster cheek pouch (P < 0.05). Both compounds had no significant effects on bradykinin-induced responses. Nanomolar concentrations of R(-)-N(6)-(2-phenylisopropyl)-adenosine [R(-)-PIA], a selective adenosine A(1) agonist, evoked significant, concentration-dependent increases in macromolecular efflux. This response was significantly attenuated by PACPX but not by DMPX. In contrast, CGS-21680, a selective adenosine A(2) agonist, increased macromolecular efflux but only at micromolar concentrations. This response was significantly attenuated by DMPX but not by PACPX. Suffusion of nitroglycerin had no significant effects on R(-)-PIA- and CGS-21680-induced responses. In addition, suffusion of N(G)-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor, had no significant effects on adenosine-induced responses. Indomethacin had no significant effects on adenosine-, R(-)-PIA-, and CGS-21680-induced increases in macromolecular efflux. Collectively, these data indicate that adenosine increases macromolecular efflux from the intact hamster cheek pouch by stimulating high-affinity adenosine A(1) receptors in a specific, nitric oxide- and prostaglandin-independent fashion.     

Chronic Effects of Xanthines on Levels of Central Receptors in Mice

1.Chronic ingestion of caffeine causes a significant increase in levels of A₁-adenosine, nicotinic and muscarinic receptors, serotonergic receptors, GABA_A receptors and L-type calcium channels in cerebral cortical membranes from mice NIH Swiss strain mice.2.Chronic theophylline and paraxanthine had effects similar to those of caffeine except that levels of L-type channels were unchanged. Chronic theobromine, a weak adenosine antagonist, and 1-isobutyl-3-methylxanthine (IBMX), a potent adenosine antagonist and phosphodiesterase inhibitor, caused only an increase in levels of A₁-adenosine receptors. A combination of chronic caffeine and IBMX had the same effects on receptors as caffeine alone. Chronic 3,7-dimethyl-1-propargylxanthine (DMPX), a somewhat selective A_2A-antagonist, caused only an increase in levels of A₁-adenosine receptors. Pentoxyfylline, an adenosine-uptake inhibitor inactive at adenosine receptors, had no effect on receptor levels or calcium channels.3.A comparison of plasma and brain levels of xanthines indicated that caffeine penetrated more readily and attained somewhat higher brain levels than theophylline or theobromine. Penetration and levels were even lower for IBMX, paraxanthine, DMPX, and pentoxyfylline.4.The results suggest that effective blockade of both A₁ and A_2A-adenosine receptors is necessary for the full spectrum of biochemical changes elicited by chronic ingestion of xanthines, such as caffeine, theophylline, and paraxanthine.     

Impaired A2A adenosine receptor/nitric oxide/VEGF signaling pathway in fetal endothelium during late- and early-onset preeclampsia

To investigate whether fetal endothelial cell proliferation and migration are modulated by the A_2A adenosine receptor (A_2AAR), nitric oxide (NO) and the vascular endothelial growth factor (VEGF) signaling pathway, we isolated human umbilical vein endothelial cells from normal pregnancy (n?=?23), preterm delivery (n?=?4), and late-onset (LOPE, n?=?10) and early-onset preeclampsia (EOPE, n?=?8). We used the non-selective adenosine receptor agonist (NECA) and the selective agonist (CGS-21680) and/or selective antagonist (ZM-241385) for A_2AAR. Also, the nitric oxide synthase (NOS) inhibitor, l-NAME, was used in co-incubation with CGS-21680. Compared to normal pregnancy, EOPE exhibited low cell proliferation and migration associated with reduced expressions of A_2AAR and VEGF and NO synthesis (i.e., total and phosphorylated serine¹¹⁷⁷ endothelial NOS and nitrite formation). In contrast, LOPE exhibited the opposite behavior in all these markers compared to normal pregnancy or EOPE. Cell proliferation and migration were increased by CGS-21680 (or NECA) in all analyzed groups (EOPE>LOPE>normal pregnancy) compared to their respective basal conditions, an effect that was associated with high NO and VEGF synthesis and blocked by ZM-241385 with significantly different IC₅₀ for each group (EOPE>LOPE>normal pregnancy). The differences seem independent of gestational age. l-NAME blocked the CGS-21680-mediated cell proliferation and migration in normal pregnancy and LOPE (IC₅₀?=?36.2?±?2.5 and 8.6?±?2.2 nM, respectively) as well as the VEGF expression in normal pregnancy. Therefore, the A_2AAR/NO/VEGF signaling pathway exhibits a pro-angiogenic effect in normal pregnancies and LOPE, whereas impairment in this pathway seems related to the reduced angiogenic capacity of the fetal endothelium in EOPE.     

Modulation of protein kinase C by adenosine: Involvement of adenosine A1 receptor-pertussis toxin sensitive nucleotide binding protein system

The objective of this study was to determine whether adenosine A₁ or A₂ receptor was responsible for the regulation of protein kinase C (PKC) in porcine coronary artery and its coupling to G-protein. Endothelium denuded arterial rings were incubated with PDBu (200nM) in the presence or absence of adenosine receptor agonists and antagonists for 1 day. Following incubation, the arterial rings were contracted with increasing concentrations of endothelin-1 (ET-1) (10^–10–10^–7M). Arteries incubated with PDBu alone failed to produce contraction in response to ET-1. On the contrary, inclusion of A₁ receptor agonist ENBA at 10^–9M in the incubation media with PDBu protected against the PDBu induced blunting of the ET-1 contractions by 50%. Incubation with ENBA alone increased ET-1 dependent contractions by about 2 fold. Inclusion of A₁ receptor antagonist, N0861 at 10^–6 M along with PDBu and ENBA, completely blocked the protective effect of ENBA against the PDBu induced attenuation of ET-1 contractions. N0861 also completely blocked the increase in ET-1 contractions in the arterial rings incubated with ENBA alone. Another A₁ receptor antagonist DPCPX also produced similar results as N0861. On the contrary, arterial rings incubated with relatively specific A₂ receptor agonist CGS 21680 at 10^–4M did not produce any protection against PDBu induced blunting of the ET-1 contractions. Incubation with CGS 21680 alone also did not significantly alter the ET-1 contractions. Interestingly, inclusion of A₂ receptor antagonist DMPX at 10^–4M in the incubation media along with CGS 21680 mimicked the effects of ENBA alone i.e. produced protection against PDBu and enhanced ET-1 contractions. Incubation of the arteries with ENBA alone caused an accumulation of PKC levels, whereas, incubation with CGS 21680 had no significant effect on PKC levels. To study the coupling of adenosine receptor with G-protein, the tissue was incubated for one day with cholera (CT) or pertussis toxin (PT) in the presence or absence or ENBA and PDBu as described above. Incubation with PT blocked the protective effect of ENBA against PDBu as well as the elevation of ET-1 response when incubated with ENBA alone. On the contrary, incubation with CT did not produce any significant effect on ENBA responses. These results indicate that PKC is modulated by adenosine via A₁ adenosine receptors and through a PT sensitive G-protein.This work was supported by National Heart, Lung and Blood Institute Grant HL-27339.     

Serotonin 5-HT1A Receptor Activation Increases Cyclic AMP Formation in the Rat Hippocampus In Vivo

Abstract: In vivo microdialysis was used to examine the efflux of cyclic AMP (cAMP) into the extracellular fluid of the ventral hippocampus in the freely moving rat. The changes in extracellular cAMP concentration were monitored in response to forskolin and the serotonin 5-HT_1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). The basal level of hippocampal extracellular cAMP was 2.3 ± 0.2 pmol/ml (n = 6), after a 3-h postsur- gery stabilisation period. Perfusion of forskolin (100 μM) through the probe for 30 min significantly increased the efflux of cAMP, which returned to baseline levels within 90 min. 8-OH-DPAT (0.3 mg/kg s.c.) also significantly increased cAMP efflux, whereas a similar volume of saline had no effect. Desensitisation of the 8-OH-DPAT-induced increase in cAMP efflux was observed following a second administration of 8-OH-DPAT after a 4-h interval. Administration of 8-OH-DPAT did not alter the efflux of cAMP when forskolin was perfused through the probe. Pretreatment with WAY 100135 [N-tert-butyl 3–4-(2-methoxyphenyl)piperazine-1 -yl-2-phenylpropanamide dihydrochloride] (5 mg/kg s.c.), a specific 5-HT_1A receptor antagonist, prevented the 8-OH-DPAT-induced increase in cAMP efflux. The data indicate that the 8-OH-DPAT-induced increase in cAMP efflux in vivo is mediated by a 5-HT_1A receptor.     

Inhibition of Striatal GABA Release by the Adenosine A2a Receptor Is Not Mediated by Increases in Cyclic AMP

Abstract: The adenosine A_2a receptor inhibition of potassium (15 m M )-evoked GABA release from striatal nerve terminals has been examined. High extracellular calcium concentrations (4 m M ) reduced the effect of the A_2a receptor agonist CGS-21680 (1 n M ). CGS-21680 inhibited GABA release in the presence of the L-type calcium channel blocker nifedipine, which itself inhibited evoked GABA release (by 16 ± 4%). ω-Conotoxin inhibited the evoked release by 45 ± 4% and prevented the action of CGS-21680. Forskolin and 8-bromo cyclic AMP both stimulated evoked GABA release at low concentrations, but at higher concentrations they abolished the inhibition by CGS-21680 without affecting the evoked release. The nonselective protein kinase inhibitor H-7 inhibited both the evoked release and the inhibition by CGS-21680, whereas the selective protein kinase A and G inhibitor HA-1004 had no effect on either evoked release or the action of CGS-21680. Pretreatment with pertussis toxin did not affect the A_2a receptor-mediated inhibition. Therefore, the effect of A_2a receptor stimulation was not mediated by protein kinases A or G but was inhibited by elevated cyclic AMP levels and mimicked by inhibitors of the N-type calcium channel and protein kinase C.     

Regulation of Glial Na+/K+-ATPase by Serotonin: Identification of Participating Receptors

The purpose of the present study was the characterization of the receptors participating in the regulatory mechanism of glial Na⁺/K⁺-ATPase by serotonin (5-HT) in rat brain. The activity of the Na⁺ pump was measured in four brain regions after incubation with various concentrations of serotoninergic agonists or antagonists. A concentration-dependent increase in enzyme activity was observed with the 5-HT_1A agonist R (+)-2-dipropylamino-8-hydroxy-1,2,3, 4-tetrahydronaphthalene hydrobromide (8-OH-DPAT) in homogenates or in glial membrane enriched fractions from cerebral cortex and in hippocampus. Spiperone, a 5-HT_1A antagonist, completely inhibited the response to 8-OH-DPAT but had no effect on Na⁺/K⁺-ATPase activity in cerebellum where LSD, a 5-HT₆ agonist, elicited a dose-dependent response similar to that of 5-HT. In brainstem, a lack of reponse to 5-HT and other agonists was confirmed. Altogether, these results show that serotonin modulates glial Na⁺/K⁺-ATPase activity in the brain, apparently not through only one type of 5-HT receptor. It seems that the receptor system involved is different according to the brain region. In cerebral cortex, the response seems to be mediated by 5-HT_1A as well as in hippocampus but not in cerebellum where 5-HT₆ appears as the receptor system involved.     

Expression of A1 adenosine receptors in the developing avian retina: in vivo modulation by A2A receptors and endogenous adenosine

Little is known about the mechanisms that regulate the expression of adenosine receptors during CNS development. We demonstrate here that retinas from chick embryos injected in ovo with selective adenosine receptor ligands show changes in A1 receptor expression after 48 h. Exposure to A1 agonist N⁶‐cyclohexyladenosine (CHA) or antagonist 8‐Cyclopentyl‐1, 3‐dipropylxanthine (DPCPX) reduced or increased, respectively, A1 receptor protein and [³H]DPCPX binding, but together, CHA+DPCPX had no effect. Interestingly, treatment with A_2A agonist 3‐[4‐[2‐[[6‐amino‐9‐[(2R,3R,4S,5S)‐5‐(ethylcarbamoyl)‐3,4‐dihydroxy‐oxolan‐2‐yl]purin‐2‐yl]amino] ethyl]phenyl] propanoic acid (CGS21680) increased A1 receptor protein and [³H]DPCPX binding, and reduced A_2A receptors. The A_2A antagonists 7‐(2‐phenylethyl)‐5‐amino‐2‐(2‐furyl)‐pyrazolo‐[4,3‐e]‐1,2,4‐trizolo[1,5‐c] pyrimidine (SCH58261) and 4‐(2‐[7‐amino‐2‐[2‐furyl][1,2,4]triazolo[2,3‐a][1,3,5]triazo‐5‐yl‐amino]ethyl)phenol (ZM241385) had opposite effects on A1 receptor expression. Exposure to CGS21680 + CHA did not change A1 receptor levels, whereas CHA + ZM241385 or CGS21680 + DPCPX had no synergic effect. The blockade of adenosine transporter with S‐(4‐nitrobenzyl)‐6‐thioinosine (NBMPR) also reduced [³H]DPCPX binding, an effect blocked by DPCPX, but not enhanced by ZM241385. [³H]DPCPX binding kinetics showed that treatment with CHA reduced and CGS21680 increased the Bmax, but did not affect Kd values. CHA, DPCPX, CGS21680, and ZM241385 had no effect on A1 receptor mRNA. These data demonstrated an in vivo regulation of A1 receptor expression by endogenous adenosine or long‐term treatment with A1 and A_2A receptors modulators.     

Adenosine A<Subscript>2A</Subscript> receptor regulates expression of vascular endothelial growth factor in feto-placental endothelium from normal and late-onset pre-eclamptic pregnancies

We aim to investigate whether A_2A/nitric oxide-mediated regulation of vascular endothelial growth factor (VEGF) expression is impaired in feto-placental endothelial cells from late-onset pre-eclampsia. Cultures of human umbilical vein endothelial cells (HUVECs) and human placental microvascular endothelial cells (hPMECs) from normal and pre-eclamptic pregnancies were used. Assays by using small interference RNA (siRNA) for A_2A were performed, and transfected cells were used for estimation of messenger RNA (mRNA) levels of VEGF, as well as for cell proliferation and angiogenesis in vitro. CGS-21680 (A_2A agonist, 24 h) increases HUVEC and hPMEC proliferation in a dose response manner. Furthermore, similar to CGS-21680, the nitric oxide donor, S-nitroso-N-acetyl-penicillamine oxide (SNAP), increased cell proliferation in a dose response manner (logEC₅₀ 10^?9.2 M). In hPMEC, CGS-21680 increased VEGF protein levels in both normal (～1.5-fold) and pre-eclamptic pregnancies (～1.2-fold), an effect blocked by the A_2A antagonist, ZM-241385 (10^?5 M) and the inhibitor of NO synthase, N _ω-nitro-L-arginine methyl ester hydrochloride (L-NAME). Subsequently, SNAP partially recovered cell proliferation and in vitro angiogenesis capacity of cells from normal pregnancies exposed to siRNA for A_2A. CGS-21680 also increased (～1.5-fold) the level of VEGF mRNA in HUVEC from normal pregnancies, but not in pre-eclampsia. Additionally, transfection with siRNA for A_2A decrease (～30 %) the level of mRNA for VEGF in normal pregnancy compared to untransfected cells, an effect partially reversed by co-incubation with SNAP. The A_2A-NO-VEGF pathway is present in endothelium from microcirculation and macrocirculation in both normal and pre-eclamptic pregnancies. However, NO signaling pathway seems to be impaired in HUVEC from pre-eclampsia.     

Evidence for Presynaptic Adenosine A2a Receptors Associated with Norepinephrine Release and Their Desensitization in the Rat Nucleus Tractus Solitarius

Abstract: Rat medullary brain segments containing primarily nucleus tractus solitarius (NTS) were used for superfusion studies of evoked transmitter release and for isotherm receptor binding assays. Isotherm binding assays with [³H]CGS-21680 on membranes prepared from NTS tissue blocks indicated a single high-affinity binding site with a K_D of 5.1 ± 1.4 nM and a B_max of 20.6 ± 2.4 fmol/mg of protein. The binding density for [³H]CGS-21680 on NTS membranes was 23 times less than comparable binding on membranes from striatal tissue. Electrically stimulated (1 min at 25 mA, 2 ms, 3 Hz) release of [³H]norepinephrine ([³H]NE) from 400-µm-thick NTS tissue slices resulted in an S₂/S₁ ratio of 0.96 ± 0.02. Superfusion of single tissue slices with 0.1–100 nM CGS-21680, a selective adenosine A_2a receptor agonist, for 5 min before the S₂ stimulus produced a significant concentration-dependent increase in the S₂/S₁ fractional release ratio that was maximal (31.3% increase) at 1.0 nM. However, superfusion of tissue slices with CGS-21680 over the same concentration range for 20 min before the S₂ stimulus did not alter the S₂/S₁ ratio significantly from control release ratios. The augmented release of [³H]NE mediated by 1.0 nM CGS-21680 with a 5-min tissue exposure was abolished by 1.0 and 10 nM CGS-15943 as well as by 100 nM 8-(3-chlorostyryl)caffeine, both A_2a receptor antagonists, but not by 1.0 nM 8-cyclopentyl-1,3-dipropylxanthine, the A₁ receptor antagonist. Taken together, these results suggest that CGS-21680 augmented the evoked release of [³H]NE in the NTS via activation of presynaptic A_2a receptors within the same concentration range as the binding affinity observed for [³H]CGS-21680. It was also apparent that this population of presynaptic adenosine A_2a receptors in the NTS desensitized within 20 min because the augmenting action of CGS-21680 on evoked transmitter release was not evident at the longer interval.     

Molecular modeling of a PAMAM-CGS21680 dendrimer bound to an A2A adenosine receptor homodimer

The theoretical possibility of bivalent binding of a dendrimer, covalently appended with multiple copies of a small ligand, to a homodimer of a G protein-coupled receptor was investigated with a molecular modeling approach. A molecular model was constructed of a third generation (G3) poly(amidoamine) (PAMAM) dendrimer condensed with multiple copies of the potent A_2A adenosine receptor agonist CGS21680. The dendrimer was bound to an A_2A adenosine receptor homodimer. Two units of the nucleoside CGS21680 could occupy the A_2A receptor homodimer simultaneously. The binding mode of CGS21680 moieties linked to the PAMAM dendrimer and docked to the A_2A receptor was found to be similar to the binding mode of a monomeric CGS21680 ligand.     

Specific Activation of A3, A2A and A1 Adenosine Receptors in CD73-Knockout Mice Affects B16F10 Melanoma Growth,Neovascularization, Angiogenesis and Macrophage Infiltration

CD73 (ecto-5''-nucleotidase), a cell surface enzyme hydrolyzing AMP to adenosine, was lately demonstrated to play a direct role in tumor progression including regulation of tumor vascularization. It was also shown to stimulate tumor macrophage infiltration. Interstitial adenosine, accumulating in solid tumors due to CD73 enzymatic activity, is recognized as a main mediator regulating the production of pro- and anti-angiogenic factors, but the engagement of specific adenosine receptors in tumor progression in vivo is still poorly researched. We have analyzed the role of high affinity adenosine receptors A₁, A_2A, and A₃ in B16F10 melanoma progression using specific agonists (CCPA, CGS-21680 and IB-MECA, respectively). We limited endogenous extracellular adenosine background using CD73 knockout mice treated with CD73 chemical inhibitor, AOPCP (adenosine α,β-methylene 5’-diphosphate). Activation of any adenosine receptor significantly inhibited B16F10 melanoma growth but only at its early stage. At 14th day of growth, the decrease in tumor neovascularization and MAPK pathway activation induced by CD73 depletion was reversed by all agonists. Activation of A₁AR primarily increased angiogenic activation measured by expression of VEGF-R2 on tumor blood vessels. However, mainly A₃AR activation increased both the microvessel density and expression of pro-angiogenic factors. All agonists induced significant increase in macrophage tumor infiltration, with IB-MECA being most effective. This effect was accompanied by substantial changes in cytokines regulating macrophage polarization between pro-inflammatory and pro-angiogenic phenotype. Our results demonstrate an evidence that each of the analyzed receptors has a specific role in the stimulation of tumor angiogenesis and confirm significantly more multifaceted role of adenosine in its regulation than was already observed. They also reveal previously unexplored consequences to extracellular adenosine signaling depletion in recently proposed anti-CD73 cancer therapy.     

Involvement of adenosine A1 receptors in antitussive effect in mice

The effects of N⁶-cyclohexyladenosine, a selective adenosine A₁ receptor agonist, on the capsaicin-induced cough reflex in mice were examined. I.c.v. administration of N⁶-cyclohexyladenosine in doses that ranged from 0.03 to 0.3 nmol decreased the number of coughs in a dose-dependent manner. Pretreatment with 8-cyclopentyl-1,3-theophylline, a selective adenosine A₁ receptor antagonist, significantly reduced the antitussive effect of N⁶-cyclohexyladenosine. On the other hand, CGS21680 (0.3 and 1 nmol, i.c.v.), a selective adenosine A₂ receptor agonist, had no significant effect on the number of capsaicin-induced coughs. These data suggest that adenosine A₁ agonist may have a marked antitussive effect in mice.     

Rapid Desensitization of Serotonin 5-HT2C Receptor-Stimulated Intracellular Calcium Mobilization in CHO Cells Transfected with Cloned Human 5-HT2C Receptors

Abstract: Serotonin 5-HT_2C receptor-mediated intracellular Ca²⁺ mobilization was investigated in Chinese hamster ovary (CHO) cells transfected with 5-HT_2C receptors. Fura-2 acetoxymethyl ester was used to investigate the regulation of 5-HT_2C receptor function. CHO cells, transfected with a cDNA clone for the 5-HT_2C receptor, expressed 287 fmol/mg of the receptor protein as determined by mianserin-sensitive [³H]mesulergine binding (K_D = 0.49 nM). The addition of 5-HT mobilized intracellular Ca²⁺ in a dose-dependent fashion, ranging from a basal level of 99 ± 1.8 up to 379 ± 18 nM, with an EC₅₀ value for 5-HT of 0.029 µM. Exposure to 5-HT, 1-(3-chlorophenyl)piperazine dihydrochloride (a 5-HT_2C agonist), and 1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane (a 5-HT_2C and 5-HT_2A agonist) resulted in increased intracellular Ca²⁺ levels. Mianserin, mesulergine, ritanserin, and ketanserin each blocked 5-HT-mediated intracellular Ca²⁺ mobilization more effectively than spiperone. The receptor was rapidly desensitized by preexposure to 5-HT in a time- and concentration-dependent manner. Mezerein and phorbol 12-myristate 13-acetate, protein kinase C activators, weakly inhibited the intracellular Ca²⁺ mobilization induced by 10 µM 5-HT. Furthermore, the protein kinase C inhibitor H-7 partially prevented the protein kinase C activator-induced inhibition of the 5-HT-mediated increase in intracellular Ca²⁺ concentration. The desensitization induced by pretreatment with 5-HT was blocked by W-7, added in conjunction with 5-HT, and partially inhibited by W-5, a nonselective inhibitor of protein kinases and weak analogue of W-7. Therefore, the 5-HT_2C receptor may be connected with protein kinase C and calcium/calmodulin turnover. These results suggest that 5-HT_2C receptor activation mobilizes Ca²⁺ in CHO cells and that the acute desensitization of the receptor may be due to calmodulin kinase-mediated feedback.     

The role of various calcium and potassium channels in the regulation of somatodendritic serotonin release

We prepared slices from midbrain containing the raphe nuclei and from hippocampus of rats. The brain slices were loaded with [³H]serotonin and superfused in order to measure the release of radioactivity at rest and in response to electrical stimulation. No difference was observed in the resting and stimulated fractional release of tritium in the somatodendritic and axon terminal parts of serotonergic neurons. The selective 5-HT_1A receptor agonist 8-OH-DPAT decreased the electrically induced tritium effux from raphe nuclei slices preloaded with [³H]serotonin, and this inhibition was reversed by 5-HT_1A receptor antagonist (+)WAY-100135. The 5-HT_1B receptor agonist CGS-12066B but not 8-OH-DPAT, inhibited the stimulation-evoked tritium efflux from hippocampal slices after labeling with [³H]serotonin. The electrical stimulation-evoked tritium efflux in raphe nuclei slices incubate with [³H]serotonin was completely external Ca²⁺-dependent, and omega-conotoxin GVIA and Cd²⁺, but not diltiazem, inhibited the tritium overflow. In raphe nuclei slices 4-aminopyridine enhanced the electrical stimulation-induced trititum release in a concentration-dependent manner. The inhibition of tritium efflux by 8-OH-DPAT was abolished with 4-aminopyridine. Glibenclamide or tolbutamide proved to be ineffective. These data indicate that (1) different 5-HT receptor subtypes (5-HT_1A and 5-HT_1B) regulate dendritic and axon terminal 5-HT release; (2) serotonin release from the dendrites may be regulated by the voltage-sensitive N-type Ca²⁺ channels; (3) the 5-HT_1A receptor-mediated inhibition of serotonin release may be due to opening of voltage-sensitive K⁺ channels.