Inhibitory Adenosine A1 Receptors Research Articles

The ADORA1 mutation linked to early-onset Parkinson’s disease alters adenosine A1-A2A receptor heteromer formation and function

Adenosine modulates neurotransmission through inhibitory adenosine A1 receptors (A1Rs) and stimulatory A2A receptors (A2ARs). These G protein-coupled receptors are involved in motor function and related to neurodegenerative diseases such as Parkinson’s disease (PD). An autosomal-recessive mutation (G2797.44S) within the transmembrane helix (TM) 7 of A1R (A1RG279S) has been associated with the development of early onset PD (EOPD). Here, we aimed at investigating the impact of this mutation on the structure and function of the A1R and the A1R-A2AR heteromer. Our results revealed that the G2797.44S mutation does not alter A1R expression, ligand binding, constitutive activity or coupling to transducer proteins (Gαi, Gαq, Gα12/13, Gαs, β-arrestin2 and GRK2) in transfected HEK-293 T cells. However, A1RG279S weakened the ability of A1R to heteromerize with A2AR, as shown in a NanoBiT assay, which led to the disappearance of the heteromerization-dependent negative allosteric modulation that A1R imposes on the constitutive activity and agonist-induced activation of the A2AR. Molecular dynamic simulations allowed to propose an indirect mechanism by which the G2797.44S mutation in TM 7 of A1R weakens the TM 5/6 interface of the A1R-A2AR heteromer. Therefore, it is demonstrated that a PD linked ADORA1 mutation is associated with dysfunction of adenosine receptor heteromerization. We postulate that a hyperglutamatergic state secondary to increased constitutive activity and sensitivity to adenosine of A2AR not forming heteromers with A1R could represent a main pathogenetic mechanism of the EOPD associated with the G2797.44S ADORA1 mutation.

Brain Iron Deficiency Changes the Stoichiometry of Adenosine Receptor Subtypes in Cortico-Striatal Terminals: Implications for Restless Legs Syndrome.

Brain iron deficiency (BID) constitutes a primary pathophysiological mechanism in restless legs syndrome (RLS). BID in rodents has been widely used as an animal model of RLS, since it recapitulates key neurochemical changes reported in RLS patients and shows an RLS-like behavioral phenotype. Previous studies with the BID-rodent model of RLS demonstrated increased sensitivity of cortical pyramidal cells to release glutamate from their striatal nerve terminals driving striatal circuits, a correlative finding of the cortical motor hyperexcitability of RLS patients. It was also found that BID in rodents leads to changes in the adenosinergic system, a downregulation of the inhibitory adenosine A1 receptors (A1Rs) and upregulation of the excitatory adenosine A2A receptors (A2ARs). It was then hypothesized, but not proven, that the BID-induced increased sensitivity of cortico-striatal glutamatergic terminals could be induced by a change in A1R/A2AR stoichiometry in favor of A2ARs. Here, we used a newly developed FACS-based synaptometric analysis to compare the relative abundance on A1Rs and A2ARs in cortico-striatal and thalamo-striatal glutamatergic terminals (labeled with vesicular glutamate transporters VGLUT1 and VGLUT2, respectively) of control and BID rats. It could be demonstrated that BID (determined by measuring transferrin receptor density in the brain) is associated with a selective decrease in the A1R/A2AR ratio in VGLUT1 positive-striatal terminals.

Adenosine A1 and A2A Receptors in the Brain: Current Research and Their Role in Neurodegeneration.

The inhibitory adenosine A1 receptor (A1R) and excitatory A2A receptor (A2AR) are predominantly expressed in the brain. Whereas the A2AR has been implicated in normal aging and enhancing neurotoxicity in multiple neurodegenerative diseases, the inhibitory A1R has traditionally been ascribed to have a neuroprotective function in various brain insults. This review provides a summary of the emerging role of prolonged A1R signaling and its potential cross-talk with A2AR in the cellular basis for increased neurotoxicity in neurodegenerative disorders. This A1R signaling enhances A2AR-mediated neurodegeneration, and provides a platform for future development of neuroprotective agents in stroke, Parkinson’s disease and epilepsy.

High sucrose consumption induces memory impairment in rats associated with electrophysiological modifications but not with metabolic changes in the hippocampus

High sugar consumption is a risk factor for metabolic disturbances leading to memory impairment. Thus, rats subject to high sucrose intake (HSu) develop a metabolic syndrome and display memory deficits. We now investigated if these HSu-induced memory deficits were associated with metabolic and electrophysiological alterations in the hippocampus. Male Wistar rats were submitted for 9weeks to a sucrose-rich diet (35% sucrose solution) and subsequently to a battery of behavioral tests; after sacrifice, their hippocampi were collected for ex vivo high-resolution magic angle spinning (HRMAS) metabolic characterization and electrophysiological extracellular recordings in slices. HSu rats displayed a decreased memory performance (object displacement and novel object recognition tasks) and helpless behavior (forced swimming test), without altered locomotion (open field). HRMAS analysis indicated a similar hippocampal metabolic profile of HSu and control rats. HSu rats also displayed no change of synaptic transmission and plasticity (long-term potentiation) in hippocampal Schaffer fibers-CA1 pyramid synapses, but had decreased amplitude of long-term depression in the temporoammonic (TA) pathway. Furthermore, HSu rats had an increased density of inhibitory adenosine A1 receptors (A1R), that translated into a greater potency of A1R in Schaffer fiber synapses, but not in the TA pathway, whereas the endogenous activation of A1R in HSu rats was preserved in the TA pathway but abolished in Schaffer fiber synapses. These results suggest that HSu triggers a hippocampal-dependent memory impairment that is not associated with altered hippocampal metabolism but is probably related to modified synaptic plasticity in hippocampal TA synapses

Lack of endogenous adenosine tonus on sympathetic neurotransmission in spontaneously hypertensive rat mesenteric artery.

BackgroundIncreased sympathetic activity has been implicated in hypertension. Adenosine has been shown to play a role in blood flow regulation. In the present study, the endogenous adenosine neuromodulatory role, in mesenteric arteries from normotensive and spontaneously hypertensive rats, was investigated.Methods and ResultsThe role of endogenous adenosine in sympathetic neurotransmission was studied using electrically-evoked [3H]-noradrenaline release experiments. Purine content was determined by HPLC with fluorescence detection. Localization of adenosine A1 or A2A receptors in adventitia of mesenteric arteries was investigated by Laser Scanning Confocal Microscopy. Results indicate a higher electrically-evoked noradrenaline release from hypertensive mesenteric arteries. The tonic inhibitory modulation of noradrenaline release is mediated by adenosine A1 receptors and is lacking in arteries from hypertensive animals, despite their purine levels being higher comparatively to those determined in normotensive ones. Tonic facilitatory adenosine A2A receptor-mediated effects were absent in arteries from both strains. Immunohistochemistry revealed an adenosine A1 receptors redistribution from sympathetic fibers to Schwann cells, in adventitia of hypertensive mesenteric arteries which can explain, at least in part, the absence of effects observed for these receptors.ConclusionData highlight the role of purines in hypertension revealing that an increase in sympathetic activity in hypertensive arteries is occurring due to a higher noradrenaline/ATP release from sympathetic nerves and the loss of endogenous adenosine inhibitory tonus. The observed nerve-to-glial redistribution of inhibitory adenosine A1 receptors in hypertensive arteries may explain the latter effect.

Sa1742 Beta-Nicotinamide Adenine Dinucleotide (Beta-NAD) Acts At Inhibitory Adenosine A1 Receptors Expressed by Neurons in the Enteric Neural Circuits That Control Mucosal Secretion in Guinea Pig and Human Small Intestine

Sa1742 Beta-Nicotinamide Adenine Dinucleotide (Beta-NAD) Acts At Inhibitory Adenosine A1 Receptors Expressed by Neurons in the Enteric Neural Circuits That Control Mucosal Secretion in Guinea Pig and Human Small Intestine

Putative depolarisation-induced retrograde signalling accelerates the repeated hypoxic depression of excitatory synaptic transmission in area CA1 of rat hippocampus via group I metabotropic glutamate receptors

Excitatory synaptic transmission in area CA1 of the mammalian hippocampus is rapidly depressed during hypoxia. The depression is largely attributable to an increase in extracellular adenosine and activation of inhibitory adenosine A1 receptors on presynaptic glutamatergic terminals. However, sequential exposure to hypoxia results in a slower subsequent hypoxic depression of excitatory synaptic transmission, a phenomenon we have previously ascribed to a reduction in the release of extracellular adenosine. In the present study we show that this delayed depression of excitatory postsynaptic currents (EPSCs) to repeated hypoxia can be reversed by a period of postsynaptic depolarisation delivered to an individual CA1 neuron, under whole-cell voltage clamp, between two periods of hypoxia. The depolarisation-induced acceleration of the hypoxic depression of the EPSC is dependent upon postsynaptic Ca2+ influx, the activation of PKC and is blocked by intracellular application of GDP-β-S and N-ethylmaleimide (NEM), inhibitors of membrane fusion events. In addition, the acceleration of the hypoxic depression of the EPSC was prevented by the GI mGluR antagonist AIDA, but not by the CB1 cannabinoid receptor antagonist AM251. Our results suggest a process initiated in the postsynaptic cell that can influence glutamate release during subsequent metabolic stress. This may reflect a novel neuroprotective strategy potentially involving retrograde release of adenosine and/or glutamate.

Intracellular ATP Influences Synaptic Plasticity in Area CA1 of Rat Hippocampus via Metabolism to Adenosine and Activity-Dependent Activation of Adenosine A1Receptors

The extent to which brain slices reflect the energetic status of the in vivo brain has been a subject of debate. We addressed this issue to investigate the recovery of energetic parameters and adenine nucleotides in rat hippocampal slices and the influence this has on synaptic transmission and plasticity. We show that, although adenine nucleotide levels recover appreciably within 10 min of incubation, it takes 3 h for a full recovery of the energy charge (to ≥ 0.93) and that incubation of brain slices at 34°C results in a significantly higher ATP/AMP ratio and a threefold lower activity of AMP-activated protein kinase compared with slices incubated at room temperature. Supplementation of artificial CSF with d-ribose and adenine (Rib/Ade) increased the total adenine nucleotide pool of brain slices, which, when corrected for the influence of the dead cut edges, closely approached in vivo values. Rib/Ade did not affect basal synaptic transmission or paired-pulse facilitation but did inhibit long-term potentiation (LTP) induced by tetanic or weak theta-burst stimulation. This decrease in LTP was reversed by strong theta-burst stimulation or antagonizing the inhibitory adenosine A(1) receptor suggesting that the elevated tissue ATP levels had resulted in greater activity-dependent adenosine release during LTP induction. This was confirmed by direct measurement of adenosine release with adenosine biosensors. These observations provide new insight into the recovery of adenine nucleotides after slice preparation, the sources of loss of such compounds in brain slices, the means by which to restore them, and the functional consequences of doing so.

Presynaptic facilitatory adenosine A2A receptors mediate fade induced by neuromuscular relaxants that exhibit anticholinesterase activity

1. Pancuronium, cisatracurium and vecuronium are antinicotinic agents that, in contrast with d-tubocurarine and hexamethonium, exhibit anticholinesterase activity. Pancuronium-, cisatracurium- and vecuronium-induced fade results from blockade of facilitatory nicotinic receptors on motor nerves, but fade produced by such agents also depends on the presynaptic activation of inhibitory muscarinic M2 receptors by acetylcholine released from motor nerve terminals and activation of inhibitory adenosine A1 receptors by adenosine released from motor nerves and muscles. The participation of presynaptic facilitatory A2A receptors in fade caused by pancuronium, cisatracurium and vecuronium has not yet been investigated. In the present study, we determined the effects of ZM241385, an antagonist of presynaptic facilitatory A2A receptors, on fade produced by these neuromuscular relaxants in the rat phrenic nerve-diaphragm (PND) preparation. 2. The muscles were stimulated indirectly at 75±3Hz to induce a sustained tetanizing muscular contraction. The lowest concentration at which each antinicotinic agent produced fade without modifying initial tetanic tension (presynaptic action) was determined. 3. d-Tubocurarine-induced fade occurred only at 55 nmol/L, a concentration that also reduced maximal tetanic tension (post-synaptic action). At 10 nmol/L, ZM 241385 alone did not produce fade, but it did attenuate pancuronium (0.32 μmol/L)-, cisatracurium (0.32 μmol/L)- and vecuronium (0.36 μmol/L)-induced fade. 4. The fade induced by the 'pure' antinicotinic agents d-tubocurarine (55 nmol/L) and hexamethonium (413 μmol/L) was not altered by 10 nmol/L ZM 241385, indicating that presynaptic adenosine A2A receptors play a significant role in the fade produced by antinicotinic agents when such agents have anticholinesterase activity.

Stimulation of adenosine A1and A2Areceptors by AMP in the submucosal plexus of guinea pig small intestine

Actions of adenosine 5'-monophosphate (AMP) on electrical and synaptic behavior of submucosal neurons in guinea pig small intestine were studied with "sharp" intracellular microelectrodes. Application of AMP (0.3-100 microM) evoked slowly activating depolarizing responses associated with increased excitability in 80.5% of the neurons. The responses were concentration dependent with an EC(50) of 3.5 +/- 0.5 microM. They were abolished by the adenosine A(2A) receptor antagonist ZM-241385 but not by pyridoxal-phosphate-6-azophenyl-2,4-disulfonic acid, trinitrophenyl-ATP, 8-cyclopentyl-1,3-dimethylxanthine, suramin, or MRS-12201220. The AMP-evoked responses were insensitive to AACOCF3 or ryanodine. They were reduced significantly by 1) U-73122, which is a phospholipase C inhibitor; 2) cyclopiazonic acid, which blocks the Ca(2+) pump in intraneuronal membranes; and 3) 2-aminoethoxy-diphenylborane, which is an inositol (1,4,5)-trisphosphate receptor antagonist. Inhibitors of PKC or calmodulin-dependent protein kinase also suppressed the AMP-evoked excitatory responses. Exposure to AMP suppressed fast nicotinic ionotropic postsynaptic potentials, slow metabotropic excitatory postsynaptic potentials, and slow noradrenergic inhibitory postsynaptic potentials in the submucosal plexus. Inhibition of each form of synaptic transmission reflected action at presynaptic inhibitory adenosine A(1) receptors. Slow excitatory postsynaptic potentials, which were mediated by the release of ATP and stimulation of P2Y(1) purinergic receptors in the submucosal plexus, were not suppressed by AMP. The results suggest an excitatory action of AMP at adenosine A(2A) receptors on neuronal cell bodies and presynaptic inhibitory actions mediated by adenosine A(1) receptors for most forms of neurotransmission in the submucosal plexus, with the exception of slow excitatory purinergic transmission mediated by the P2Y(1) receptor subtype.

Presynaptic regulation of cardiac norepinephrine release in ischemia.

In myocardial ischemia presynaptic regulation of norepinephrine release may be altered either by ischemic effects on presynaptic receptor signaling or by ischemia-evoked accumulation of endogenous agonists. Because presynaptic receptors are targets of several drugs. such alterations may have pharmacotherapeutic implications. We investigated the effect of brief ischemic periods on presynaptic regulation of norepinephrine release by alpha2-adrenoceptors, beta2-adrenoceptors, adenosine A1-, angiotensin AT1-, and bradykinin B2-receptors in isolated perfused rat hearts. Exocytotic norepinephrine release was evoked by electrical field stimulation. Paired stimulations were performed to compare the pharmacologic intervention (S2) with the release under baseline conditions (S1), and the effects of receptor agonists and antagonists were compared under nonischemic and stop-flow conditions. In summary. during brief myocardial ischemia, presynaptic modulation of norepinephrine release is differentially regulated. Autoinhibitory alpha2-adrenoceptors lose their activity, whereas stimulatory beta2-adrenoceptors are sensitized. Inhibitory adenosine A1-receptors gain importance during ischemia owing to endogenous adenosine formation. Bradykinin- and angiotensin-mediated stimulation of norepinephrine release is not affected under ischemic conditions.

Age-dependent decrease in adenosine A1 receptor binding sites in the rat brain. Effect of cis unsaturated free fatty acids.

Unsaturated free fatty acids and adenosine operate two neuromodulatory systems with opposite effects on neuronal function. Here, we tested if fatty acids controlled inhibitory adenosine A1 receptors. Arachidonate (AA, 10 microM) decreased the Bmax of an A1 receptor agonist, (R)-[3H]phenylisopropyladenosine (PIA; from 812 to 267 fmol x mg(-1) protein), and antagonist, [3H]1,3-dipropyl-8-cyclopentylxanthine (DPCPX; from 994 to 311 fmol x mg(-1) protein) and decreased the Kd of [3H]PIA (from 1.20 to 0.57 nM) binding to brain membranes of young adult rats (2 months old), these effects being mimicked by other cis but not trans unsaturated or saturated fatty acids. AA (10 microM) increased the potency of the A1 receptor agonist, 2-chloroadenosine to inhibit hippocampal synaptic transmission in young adult rats (EC50 decreased from 337 to 237 nM), which may constitute a safety feedback mechanism to control AA-induced neurotoxicity. Upon aging, there were increased free fatty acid levels and a concomitant decreased density of A1 receptors. This was more marked in hippocampal nerve terminals of aged rats (24 months old) and may be the determinant factor contributing to the lower potency of 2-choloroadenosine in aged rats (EC50 = 955 nM), in spite of the decreased Kd of PIA binding upon aging. The effects of AA on A1 receptor binding were attenuated upon aging, AA being devoid of effects in aged rats. Accordingly, AA (10 microM) failed to modify the potency of 2-choloroadenosine in aged rats (EC50 = 997 nM). However, albumin, which quenches free fatty acids, increased A1 receptor density by 65% and 2-chloroadenosine potency (EC50 = 703 nM) in aged rats, suggesting that the increased fatty acids levels in aged rats may contribute to the decreased potency of A1 receptor agonists in aged rats. Also, the observed saturation of the control by AA of A1 receptors may contribute to the decreased adaptability of neuromodulation to different firing conditions in aged rats.

Histamine H(2) receptor activated chloride conductance in myenteric neurons from guinea pig small intestine.

Whole cell perforated patch-clamp methods were used to investigate ionic mechanisms underlying histamine-evoked excitatory responses in small intestinal AH-type myenteric neurons. When physiological concentrations of Na(+), Ca(2+), and Cl(-) were in the bathing medium, application of histamine significantly increased total conductance as determined by stepping to 50 mV from a holding potential of -30 mV. The current reversed at a membrane potential of -30 +/- 5 (SE) mV and current-voltage relations exhibited outward rectification. The reversal potential for the histamine-activated current was unchanged by removal of Na(+) and Ca(2+) from the bathing medium. Reduction of Cl(-) from 155 mM to 55 mM suppressed the current when the neurons were in solutions with depleted Na(+) and Ca(2+). Current-voltage curves in solutions with reduced Cl(-) were linear and the reversal potential was changed from -30 +/- 5 mV to 7 +/- 4 mV. Niflumic acid, but not anthracene-9-carboxylic acid (9-AC) nor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), suppressed the histamine-activated current. A membrane permeable analogue of cAMP evoked currents similar to those activated by histamine. A selective histamine H(2) receptor agonist (dimaprit) mimicked the action of histamine and a selective histamine H(2) receptor antagonist (cimetidine) blocked the conductance increase evoked by histamine. A selective adenosine A(1) receptor agonist (CCPA) reduced the histamine-activated current and a selective adenosine A(1) receptor antagonist (CPT) reversed the inhibitory action. The results suggest that histamine acts at histamine H(2) receptors to increase Cl(-) conductance in AH-type enteric neurons. Cyclic AMP appears to be a second messenger in the signal transduction process. Results with a selective adenosine A(1) receptor agonist and antagonist add to existing evidence for co-coupling of inhibitory adenosine A(1) receptors and histamine H(2) receptors to adenylate cyclase in AH-type enteric neurons.

Arachidonic acid potentiates the duration of the metabotropic, protein kinase C-mediated, suppression of the inhibitory adenosine A1 receptor pathway in glutamatergic nerve terminals from rat cerebral cortex

The KCl-evoked exocytotic release of glutamate from rat cerebrocortical synaptosomes is inhibited by a presynaptic adenosine A1 receptor decreasing voltage-activated Ca 2+ entry. This inhibition was transiently suppressed by (1 S,3 R)-1-aminocyclopenthyl-1,3-dicarboxylate (ACPD) but was restored within 1 min in the continued presence of the metabotropic agonist. In the presence of 2 μM arachidonic acid ACPD initiated a prolonged suppression of the adenosine-mediated inhibition persisting for at least 10 min. Arachidonic acid (20–40 pmol) was bound per mg synaptosomal protein. Prolonged ACPD-mediated phosphorylation of the protein kinase C (PKC) substrate myristoylated alanine-rich C-kinase substrate (MARCKS) was detected in the presence but not the absence of arachidonic acid, but arachidonic acid added 2 min after ACPD was ineffective. It is concluded that arachidonic acid synergistically prolongs the metabotropic glutamate receptor-mediated activation of presynaptic PKC, suppressing inhibitory receptor pathways.

Adenosine A 2a and A 2b Receptors in Cultured Fetal Chick Heart Cells

Adenosine exerts pronounced biological effects in the heart cell. The role of multiple adenosine receptor subtypes in regulating the heart cell function is not known. Ventricular cells cultured from chick embryos 14 days in ovo were used to study a novel feature of heart cell regulation by the stimulatory adenosine receptors. The inhibitory adenosine A1 receptor pathway was first inactivated by pertussis toxin treatment of the cultures, and the effects of adenosine agonists and antagonists on the heart cell contractile amplitude, measured via an opticovideo motion detection system, and on the modulation of cAMP level were determined. Adenosine and N-ethyladenosine-5'-uronic acid (NECA), capable of activating both the adenosine A2a and A2b receptors, caused a greater increase in the contractile amplitude than did the A2a-selective agonist 2-[4-(2-carboxythyl)phenylethylamino]-5'-N-ethylcarboxamidoa denosine (CGS21680). NECA caused a biphasic increase in cAMP, which became monophasic in the presence of the A2a receptor-selective antagonist 8-(3-chlorostyryl)caffeine, whereas the CGS21680-induced cAMP response was monophasic. Blocking with 8-(3-chlorostyryl)caffeine abolished most of the CGS21680-elicited contractile or cAMP response while attenuating only part of the adenosine- or NECA-stimulated responses. Blocking with the A2b-selective antagonists 1,3-diethyl-8-phenylxanthine or alloxazine caused a more pronounced inhibititon of the contractile or cAMP response by adenosine or NECA than by CGS21680. Affinity of the A2a receptor was 60-fold higher than that of the A2b receptor. These data demonstrate that a functional A2b receptor is expressed on the heart cell and is capable of mediating augmentation of cardiac myocyte contractility and that adenosine A2a and A2b receptors, with greatly different affinity, coexist and are coupled to the same functional responses. Taken together, the data suggest a novel feature of heart cell regulation, where the high-affinity A2a receptor can play an important modulatory role in the presence of a low level of adenosine, whereas the low-affinity A2b receptor becomes functionally important when the adenosine level is high.

Effects of PACAP on morphologically identified myenteric neurons in guinea pig small bowel.

Intracellular microelectrodes were used to examine the actions of pituitary adenylate cyclase-activating peptide (PACAP) on morphologically identified myenteric neurons and glial cells of the guinea pig small bowel. PACAP-27 and PACAP-38 evoked excitatory responses in 96% of after hyperpolarizing (AH)/type 2 neurons. The half-maximal concentration for PACAP-27 was 1.5 nM. The responses consisted of membrane depolarization in association with increased input resistance, suppression of hyperpolarizing afterpotentials, and repetitive spike discharge. Forskolin mimicked the action of PACAP in all AH/type 2 neurons. PACAP excited 36% of S/type 1 neurons. Most of the AH/type 2 neurons had Dogiel II morphology, whereas the S/type 1 neurons were uniaxonal with morphology characteristics of Dogiel I or filamentous neurons. No glial cells responded to PACAP. A selective A1 adenosine receptor agonist blocked the excitatory action of PACAP, and this was reversed by a selective A1 antagonist. The results suggest that excitatory PACAP receptors and inhibitory adenosine A1 receptors are linked to adenylate cyclase in AH/type 2 myenteric neurons.