Adenosine signalling at immature parallel fibre–Purkinje cell synapses in rat cerebellum

The purine adenosine is an extracellular signalling molecule involved in a large number of physiological and pathological conditions throughout the mammalian brain. However little is known about how adenosine release and its subsequent clearance change during brain development. We have combined electrophysiology and microelectrode biosensor measurements to investigate the properties of adenosine signalling at early stages of cerebellar development, when parallel fibre–Purkinje cell synapses have recently been formed (postnatal days 9–12). At this stage of development, we could detect little or no inhibitory A₁ receptor tone in basal conditions and during trains of stimuli. Addition of pharmacological agents, to inhibit adenosine clearance, had only minor effects on synaptic transmission suggesting that under basal conditions, the concentration of adenosine moving in and out of the extracellular space is small. Active adenosine release was stimulated with hypoxia and trains of electrical stimuli. Although hypoxia released significant concentrations of adenosine, the release was delayed and slow. No adenosine release could be detected following electrical stimulation in the molecular layer. In conclusion, at this stage of development, although adenosine receptors and the mechanisms of adenosine clearance are present there is very little adenosine release.     

Toxoplasma gondii tachyzoites possess an unusual plasma membrane adenosine transporter

Nucleoside transport may play a critical role in successful intracellular parasitism by Toxoplasma gondii. This protozoan is incapable of de novo purine synthesis, and must salvage purines from the host cell. We characterized purine transport by extracellular T. gondii tachyzoites, focusing on adenosine, the preferred salvage substrate. Although wild-type RH tachyzoites concentrated [³H]adenosine 1.8-fold within 30 s, approx. half of the [³H]adenosine was converted to nucleotide, consistent with the known high parasite adenosine kinase activity. Studies using an adenosine kinase deficient mutant confirmed that adenosine transport was non-concentrative. [¹⁴C]Inosine, [¹⁴C]hypoxanthine and [³H]adenine transport was also rapid and non-concentrative. Adenosine transport was inhibited by dipyridamole (IC₅₀ approx. 0.7 μM), but not nitrobenzylthioinosine (15 μM). Transport of inosine, hypoxanthine and adenine was minimally inhibited by 10 μM dipyridamole, however. Competition experiments using unlabeled nucleosides and bases demonstrated distinct inhibitor profiles for [³H]adenosine and [¹⁴C]inosine transport. These results are most consistent with a single, dipyridamole-sensitive, adenosine transporter located in the T. gondii plasma membrane. Additional permeation pathways for inosine, hypoxanthine, adenine and other purimes may also be present.     

Involvement of adenosine in depression of synaptic transmission during hypercapnia in isolated spinal cord of neonatal rats

Adenosine is one of the most important neuromodulators in the CNS, both under physiological and pathological conditions. In the isolated spinal cord of the neonatal rat in vitro, acute hypercapnic acidosis (20% CO₂, pH 6.7) reversibly depressed electrically evoked spinal reflex potentials. This depression was partially reversed by 8-cyclopentlyl-1,3-dimethylxanthine (CPT), a selective A₁ adenosine receptor antagonist. Isohydric hypercapnia (20% CO₂, pH 7.3), but not isocapnic acidosis (5% CO₂, pH 6.7), depressed the reflex potentials, which were also reversed by CPT. An ecto-5'-nucleotidase inhibitor did not affect the hypercapnic acidosis-evoked depression. An inhibitor of adenosine kinase, but not deaminase, mimicked the inhibitory effect of hypercapnic acidosis on the spinal reflex potentials. Accumulation of extracellular adenosine and inhibition of adenosine kinase activity were caused by hypercapnic acidosis and isohydric hypercapnia, but not isohydric acidosis. These results indicate that the activation of adenosine A₁ receptors is involved in the hypercapnia-evoked depression of reflex potentials in the isolated spinal cord of the neonatal rat. The inhibition of adenosine kinase activity is suggested to cause the accumulation of extracellular adenosine during hypercapnia.     

Adenosine A1 receptor-mediated presynaptic inhibition at the calyx of Held of immature rats

At the calyx of Held synapse in brainstem slices of 5- to 7-day-old (P5–7) rats, adenosine, or the type 1 adenosine (A₁) receptor agonist N ⁶-cyclopentyladenosine (CPA), inhibited excitatory postsynaptic currents (EPSCs) without affecting the amplitude of miniature EPSCs. The A₁ receptor antagonist 8-cyclopentyltheophylline (CPT) had no effect on the amplitude of EPSCs evoked at a low frequency, but significantly reduced the magnitude of synaptic depression caused by repetitive stimulation at 10 Hz, suggesting that endogenous adenosine is involved in the regulation of transmitter release. Adenosine inhibited presynaptic Ca²⁺ currents ( I _pCa) recorded directly from calyceal terminals, but had no effect on presynaptic K⁺ currents. When EPSCs were evoked by I _pCa during simultaneous pre- and postsynaptic recordings, the magnitude of the adenosine-induced inhibition of I _pCa fully explained that of EPSCs, suggesting that the presynaptic Ca²⁺ channel is the main target of A₁ receptors. Whereas the N-type Ca²⁺ channel blocker ω-conotoxin attenuated EPSCs, it had no effect on the magnitude of adenosine-induced inhibition of EPSCs. During postnatal development, in parallel with a decrease in the A₁ receptor immunoreactivity at the calyceal terminal, the inhibitory effect of adenosine became weaker. We conclude that presynaptic A₁ receptors at the immature calyx of Held synapse play a regulatory role in transmitter release during high frequency transmission, by inhibiting multiple types of presynaptic Ca²⁺ channels.     

Force generation examined by laser temperature-jumps in shortening and lengthening mammalian (rabbit psoas) muscle fibres

The modulation of synaptic transmission by presynaptic ionotropic and metabotropic receptors is an important means to control and dynamically adjust synaptic strength. Even though synaptic transmission and plasticity at the hippocampal mossy fibre synapse are tightly controlled by presynaptic receptors, little is known about the downstream signalling mechanisms and targets of the different receptor systems. In the present study, we identified the cellular signalling cascade by which adenosine modulates mossy fibre synaptic transmission. By means of electrophysiological and optical recording techniques, we found that adenosine activates presynaptic A₁ receptors and reduces Ca²⁺ influx into mossy fibre terminals. Ca²⁺ currents are directly modulated via a membrane-delimited pathway and the reduction of presynaptic Ca²⁺ influx can explain the inhibition of synaptic transmission. Specifically, we found that adenosine modulates both P/Q- and N-type presynaptic voltage-dependent Ca²⁺ channels and thereby controls transmitter release at the mossy fibre synapse.     

Extracellular levels of adenosine and its metabolites in the striatum of awake rats: inhibition of uptake and metabolism

The level of purines in the striatum of awake, freely moving rats was studied using microdialysis. The calculated extracellular concentration of adenosine and its metabolites inosine and hypoxanthine was very high immediately after implantation of the dialysis probe but decreased within 24 h to a level which remained stable for two days. Using in vitro calibration to determine the relative recovery of the dialysis probes we estimated resting levels in the striatal extracellular space to be 40, 110 and 580 nM, respectively. Inhibition of adenosine deaminase by deoxycoformycin produced a significant 1.4-fold increase in extracellular adenosine levels and a fall in inosine and hypoxanthine. A combination of three uptake blockers (dipyridamole, lidoflazine and nitrobenzylthioinosine), caused a 4.5-fold increase in extracellular adenosine levels without any change in inosine or hypoxanthine levels. After uptake inhibition deoxycoformycin did not have any significant effect. The present results show that the microdialysis technique can be used to determine levels of purines in the extracellular fluid of defined brain regions in awake animals. The high levels recorded during the first several hours after implantation may be artefactually high and reflect trauma. The results also show that adenosine levels can be altered in vivo by inhibitors of adenosine transport and adenosine deaminase. The present results indicate that the physiological adenosine level in striatal extracellular space is in the range 40-460 nM.     

Purine salvage by Tritrichomonas foetus

The anaerobic protozoon Tritrichomonas foetus was found incapable of de novo purine synthesis by its failure to incorporate radiolabeled glycine or formate into the nucleotide pool. It had, on the other hand, high activities in incorporating adenine, hypoxanthine or inosine. Radiolabel pulse-chase experiments indicated that adenine, hypoxanthine and inosine all entered the pool through conversion to IMP. The parasite contained hypoxanthine phosphoribosyl transferase, adenine deaminase and inosine phosphorylase, but no adenine phosphoribosyl transferase, inosine kinase or inosine phosphotransferase activity. Adenine and inosine had to be converted to hypoxanthine before incorporation. Adenosine was also rapidly converted to hypoxanthine in T. foetus cell-free extracts, but the presence of adenosine kinase in the parasite allowed some conversion of adenosine directly to AMP. Guanine and xanthine were directly incorporated into GMP and XMP, probably due to the guanine and xanthine phosphoribosyl transferase. There were also strong enzyme activities which convert guanosine to guanine and guanine to xanthine. A guanosine phosphotransferase was found in the 10(5) X g sedimentable fraction of T. foetus, and was capable of converting some guanosine to GMP. This network of T. foetus purine salvage suggests the importance of hypoxanthine-guanine-xanthine phosphoribosyl transferase activities in the parasite.     

Purinergic regulation of epithelial transport

Purinergic receptors are a family of ubiquitous transmembrane receptors comprising two classes, P1 and P2 receptors, which are activated by adenosine and extracellular nucleotides (i.e. ATP, ADP, UTP and UDP), respectively. These receptors play a significant role in regulating ion transport in epithelial tissues through a variety of intracellular signalling pathways. Activation of these receptors is partially dependent on ATP (or UTP) release from cells and its subsequent metabolism, and this release can be triggered by a number of stimuli, often in the setting of cellular damage. The function of P2Y receptor stimulation is primarily via signalling through the G_q/PLC-β pathway and subsequent activation of Ca²⁺-dependent ion channels. P1 signalling is complex, with each of the four P1 receptors A₁, A_2A, A_2B, and A₃ having a unique role in different epithelial tissue types. In colonic epithelium the A_2B receptor plays a prominent role in regulating Cl⁻ and water secretion. In airway epithelium, A_2B and A₁ receptors are implicated in the control of Cl⁻ and other currents. In the renal tubular epithelium, A₁, A_2A, and A₃ receptors have all been identified as playing a role in controlling the ionic composition of the lumenal fluid. Here we discuss the intracellular signalling pathways for each of these receptors in various epithelial tissues and their roles in pathophysiological conditions such as cystic fibrosis.     

Adenosine inhibition via A1 receptor of N-type Ca2+ current and peptide release from isolated neurohypophysial terminals of the rat

Effects of adenosine on voltage-gated Ca²⁺ channel currents and on arginine vasopressin (AVP) and oxytocin (OT) release from isolated neurohypophysial (NH) terminals of the rat were investigated using perforated-patch clamp recordings and hormone-specific radioimmunoassays. Adenosine, but not adenosine 5'-triphosphate (ATP), dose-dependently and reversibly inhibited the transient component of the whole-terminal Ba²⁺ currents, with an IC₅₀ of 0.875 μ m. Adenosine strongly inhibited, in a dose-dependent manner (IC₅₀= 2.67 μ m ), depolarization-triggered AVP and OT release from isolated NH terminals. Adenosine and the N-type Ca²⁺ channel blocker ω-conotoxin GVIA, but not other Ca²⁺ channel-type antagonists, inhibited the same transient component of the Ba²⁺ current. Other components such as the L-, Q- and R-type channels, however, were insensitive to adenosine. Similarly, only adenosine and ω-conotoxin GVIA were able to inhibit the same component of AVP release. A₁ receptor agonists, but not other purinoceptor-type agonists, inhibited the same transient component of the Ba²⁺ current as adenosine. Furthermore, the A₁ receptor antagonist 8-cyclopentyltheophylline (CPT), but not the A₂ receptor antagonist 3, 7-dimethyl-1-propargylxanthine (DMPGX), reversed inhibition of this current component by adenosine. The inhibition of AVP and OT release also appeared to be via the A₁ receptor, since it was reversed by CPT. We therefore conclude that adenosine, acting via A₁ receptors, specifically blocks the terminal N-type Ca²⁺ channel thus leading to inhibition of the release of both AVP and OT.     

Contribution of Adenosine to the Depression of Sympathetically Evoked Vasoconstriction induced by Systemic Hypoxia in the Rat

Previous studies have shown that systemic hypoxia evokes vasodilatation in skeletal muscle that is mediated mainly by adenosine acting on A₁ receptors, and that the vasoconstrictor effects of sympathetic nerve activity are depressed during hypoxia. The aim of the present study was to investigate the role of adenosine in this depression. In anaesthetised rats, increases in femoral vascular resistance (FVR) evoked by stimulation of the lumbar sympathetic chain with bursts of impulses at 40 or 20 Hz were greater than those evoked by continuous stimulation at 2 Hz with the same number of impulses (120) over 1 min. All of these responses were substantially reduced by infusion of adenosine or by graded systemic hypoxia (breathing 12, 10 or 8 % O₂), increases in FVR evoked by continuous stimulation at 2 Hz being most vulnerable. Blockade of A₁ receptors ameliorated the depression caused by adenosine infusion of the increase in FVR evoked by 2 Hz only and did not ameliorate the depression caused by 8 % O₂ of increases in FVR evoked by any pattern of sympathetic stimulation. A_2A receptor blockade accentuated hypoxia-induced depression of the increase in FVR evoked by burst stimulation at 40 Hz, but had no other effect. Neither A₁ nor A_2A receptor blockade affected the depression caused by hypoxia (8 % O₂) of the FVR increase evoked by noradrenaline infusion. These results indicate that endogenously released adenosine is not responsible for the depression of sympathetically evoked muscle vasoconstriction caused by systemic hypoxia; adenosine may exert a presynaptic facilitatory influence on the vasoconstrictor responses evoked by bursts at high frequency.     

The role of adenosine in the early respiratory and cardiovascular changes evoked by chronic hypoxia in the rat

Experiments were performed on anaesthetized normoxic (N) rats and chronically hypoxic rats that had been exposed to 12% O₂ for 1, 3 or 7 days (1, 3 or 7CH rats). The adenosine A₁ receptor antagonist DPCPX did not affect the resting hyperventilation of 1–7CH rats breathing 12% O₂ and increased resting heart rate (HR) in 1CH rats only. DPCPX partially restored the decreased baseline arterial pressure (ABP) and increased femoral vascular conductance (FVC) of 1 and 3CH rats, but had no effect in N or 7CH rats. DPCPX also attenuated the decrease in arterial blood pressure (ABP) and increase in FVC evoked by acute hypoxia in N and 1–7CH rats. The non-selective adenosine receptor antagonist 8-SPT had no further effect on baselines or cardiovascular responses to acute hypoxia, but attenuated the hypoxia-evoked increase in respiratory frequency in 1–7CH rats. In N, and 1 and 3CH rats, the inducible nitric oxide synthase (iNOS) inhibitor aminoguanidine had no effect on baselines or increases in FVC evoked by acetylcholine. We propose: (i) that tonically released adenosine acting on A₁ receptors reduces HR in 1CH rats and stimulates endothelial NOS in 1 and 3CH rats to decrease ABP and increase FVC, the remaining NO-dependent tonic vasodilatation being independent of iNOS activity; (ii) that in 7CH rats, tonic adenosine release has waned; (iii) that in 1–7CH rats, adenosine released by acute hypoxia stimulates A₁ but not A₂ receptors to produce muscle vasodilatation, and stimulates carotid body A₂ receptors to increase respiration.     

Systemic Hypoxia and the Depression of Synaptic Transmission in Rat Hippocampus after Carotid Artery Occlusion

The relationship between step reductions in inspired oxygen and the amplitude of evoked field excitatory postsynaptic potentials (fEPSPs) recorded from hippocampal CA1 neurons was examined in anaesthetized rats with a unilateral common carotid artery occlusion. The amplitudes of fEPSPs recorded from the hippocampus ipsilateral to the occlusion were significantly more depressed with hypoxia than were the fEPSPs recorded from the contralateral hippocampus. The adenosine A₁-selective antagonist, 8-cyclopentyl-1,3-dimethylxanthine (8-CPT), blunted the hypoxic depression of the fEPSP. Tissue partial pressure of oxygen ( P _tiss,O2) was measured in the ipsilateral and contralateral hippocampus using glass Clark-style microelectrodes. P _tiss,O2 fell to similar levels as a function of inspired oxygen in the ipsilateral and contralateral hippocampus, and in the ipsilateral hippocampus after administration of 8-CPT. Hippocampal blood flow (HBF) was measured using laser Doppler flowmetry. A decline in HBF was associated with systemic hypoxia in both hippocampi. HBF, as a function of inspired oxygen, fell significantly more in the ipsilateral than in the contralateral hippocampus. We conclude that endogenous adenosine acting at the neuronal A₁ receptor plays a major role in the depression of synaptic transmission during hypoxic ischaemia. The greater susceptibility of the fEPSP in the ipsilateral hippocampus to systemic hypoxia cannot be explained entirely by differences in P _tiss,O2 or HBF between the two hemispheres.     

Computer simulation of ischemic rat heart purine metabolism. I. Model construction.

A model is proposed for the partial depletion of the adenine nucleotide pool in the ischemic perfused rat heart which involves seven enzymes: adenylate cyclase, 3',5'-cyclic AMP phosphodiesterase, 5'-nucleotidase, adenosine kinase, adenosine deaminase, purine nucleoside phosphorylase, and inorganic pyrophosphatase. The computer implementation of this model is in terms of rate laws, several of which were obtained by a systematic least-squares fitting procedure. Depletion of the adenine nucleotide pool is initiated by the release of endogenous noradrenaline into the interstitial fluid, which results from a fall in tissue PO2, and the subsequent activation of adenylate cyclase. In this model the substrate for 5'-nucleotidase is a membrane-bound AMP pool formed by hydrolysis of extracellular fluid and functions as a vasodilator; excess adenosine is incorporated into the tissue by a "permease" with Michaelis-Menten kinetics and converted to AMP, inosine, and hypoxanthine. Alternative mechanisms, such as the deamination of AMP by adenylate deaminase and conversion of AMP to adenine by AMP pyrophosphorylase, were rejected primarily on qualitative biochemical grounds.     

Excitatory synaptic transmission in the spinal substantia gelatinosa is under an inhibitory tone of endogenous adenosine

Exogenous adenosine produces potent synaptic inhibition in spinal substantia gelatinosa (SG), a region involved in nociceptive and thermoreceptive mechanisms. To examine the possibility that endogenous adenosine tonically modulates excitatory synaptic transmission in spinal SG, whole-cell, voltage-clamp recordings were made from SG neurons in adult rat spinal cord slices. In all SG neurons sensitive to exogenous adenosine, the adenosine uptake inhibitor, NBTI, mimics adenosine's inhibitory actions on dorsal root evoked EPSCs (eEPSCs) and miniature spontaneous EPSCs (mEPSCs). These inhibitory effects were antagonized by A1 adenosine receptor antagonist, DPCPX. DPCPX also potentates eEPSCs in those SG neurons in which adenosine or adenosine A1 receptor agonists (CHA, CCPA) suppressed eEPSCs. DPCPX often increases mEPSC frequency without altering mEPSC amplitude, suggesting presynaptic action on adenosine A1 receptors. Selective A2 (DMPX) and A2a (ZM 241385) adenosine receptor antagonists had no or minimal effects upon either eEPSCs or mEPSCs. The adenosine degrading enzyme, adenosine deaminase, mimicked the effects of DPCPX on the mEPSC frequency. We conclude that the excitatory synaptic transmission in the spinal SG is under an inhibitory tone of endogenous adenosine through the activation of A1 receptors. The present results suggested that the background activity of A1 receptors in the spinal SG might be contributed to setting the physiological “noceceptive thresholds”.     

Enhanced G protein-dependent modulation of excitatory synaptic transmission in the cerebellum of the Ca2+ channel-mutant mouse, tottering

Tottering , a mouse model for absence epilepsy and cerebellar ataxia, carries a mutation in the gene encoding class A (P/Q-type) Ca²⁺ channels, the dominant exocytotic Ca²⁺ channel at most synapses in the mammalian central nervous system. Comparing tottering to wild-type mice, we have studied glutamatergic transmission between parallel fibres and Purkinje cells in cerebellar slices. Results from biochemical assays and electrical field recordings demonstrate that glutamate release from parallel fibre terminals of the tottering mouse is controlled largely by class B Ca²⁺ channels (N-type), in contrast to the P/Q-channels that dominate release from wild-type terminals. Since N-channels, in a variety of assays, are more effectively inhibited by G proteins than are P/Q-channels, we tested whether synaptic transmission between parallel fibres and Purkinje cells in tottering mice was more susceptible to inhibitory modulation by G protein-coupled receptors than in their wild-type counterparts. GABA_B receptors and α₂-adrenergic receptors (activated by bath application of transmitters) produced a three- to fivefold more potent inhibition of transmission in tottering than in wild-type synapses. This increased modulation is likely to be important for cerebellar transmission in vivo , since heterosynaptic depression, produced by activating GABAergic interneurones, greatly prolonged GABA_B receptor-mediated presynaptic inhibition in tottering as compared to wild-type slices. We propose that this enhanced modulation shifts the balance of synaptic input to Purkinje cells in favour of inhibition, reducing Purkinje cell output from the cerebellum, and may contribute to the aberrant motor phenotype that is characteristic of this mutant animal.     

GABAB receptor activation desensitizes postsynaptic GABAB and A1 adenosine responses in rat hippocampal neurones

Whole-cell recordings of EPSCs and G-protein-activated inwardly rectifying (GIRK) currents were made from cultured hippocampal neurones to determine the effect of long-term agonist treatment on the presynaptic and postsynaptic responses mediated by GABA_B receptors (GABA_BRs). GABA_BR-mediated presynaptic inhibition was unaffected by agonist (baclofen) treatment for up to 48 h, and was desensitized by about one-half after 96 h. In contrast, GABA_BR-mediated GIRK currents were desensitized by a similar amount after only 2 h of agonist treatment. In addition, presynaptic inhibition mediated by A₁ adenosine receptors (A₁Rs) was unaffected by prolonged GABA_BR activation, whereas A₁R-mediated GIRK currents were desensitized. Desensitization of postsynaptic GABA_BR and A₁R responses was blocked by the GABA_BR antagonist (1-(S)-3,4-dichlorophenylethyl)amino-2-(S) hydroxypropyl-p-benzyl-phosphonic acid (CGP 55845A), but not by the A₁R antagonist cyclopentyldipropylxanthine (DPCPX). GIRK current amplitude could be partially restored after baclofen treatment by either coapplication of baclofen and adenosine, or intracellular infusion of the non-hydrolysable GTP analog 5'-guanylylimidodiphosphate (Gpp(NH)p). Short-term (4-24 h) baclofen treatment also significantly desensitized the inhibition of postsynaptic voltage-gated calcium channels by activation of GABA_BRs or A₁Rs. These results show that responses mediated by GABA_BRs and A₁Rs desensitize differently in presynaptic and postsynaptic compartments, and demonstrate the heterologous desensitization of postsynaptic A1R responses.     

Purine metabolism by the avian malarial parasite Plasmodium lophurae

Extracts of normal duckling erythrocytes catabolized AMP to IMP, inosine and hypoxanthine; adenosine and adenine were not formed from AMP. When erythrocyte-free Plasmodium lophurae, prepared by antibody lysis, were incubated in the presence of [¹⁴C]hypoxanthine approximately 60% of the label was recovered as purine nucleotides and there was no evidence for extracellular alteration of added hypoxanthine. However, when adenosine was added to suspensions of antibody- or saponin-prepared parasites extensive conversion to inosine and hypoxanthine occurred. This conversion was found to be the result of parasite lysis with release of cytosolic purine salvage pathway enzymes; plasmodial surface membrane ecto-enzymes were not responsible for adenosine catabolism. It appears that in vivo the intracellular plasmodium utilizes the normal erythrocytic process of purine turnover to avail itself of hypoxanthine, the red cell's end-product, and at the same time the parasite avoids direct competition for adenosine essential to erythrocyte survival. Since the blood plasma of infected ducklings contained increased amounts of hypoxanthine it is possible that P. lophurae also utilizes this as a purine source.     

A1 and A2A receptor activation by endogenous adenosine is required for VIP enhancement of K+ -evoked [3H]-GABA release from rat hippocampal nerve terminals

Vasoactive intestinal peptide (VIP) modulates GABA release from hippocampal nerve terminals and enhances hippocampal synaptic transmission through a pathway dependent on GABAergic transmission. Since VIP modulation of hippocampal synaptic transmission is dependent on the tonic actions of adenosine we investigated if endogenous adenosine could influence VIP enhancement of GABA release from isolated hippocampal nerve endings, and which adenosine receptors could be mediating this influence. When extracellular endogenous adenosine was removed using adenosine deaminase (ADA, 1 U/ml), the enhancement (57.2 ± 3.7%) caused by VIP on GABA release was prevented. Blockade of adenosine A₁ receptors with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 10 nM) or of A_2A receptors with ZM241385 (50 nM) abolished the effect of VIP. In the presence of ADA, selective A_2A receptor-activation with CGS21680 (10 nM) readmitted most of the enhancement caused by VIP on GABA release (50.7 ± 5.3%). Also in the presence of ADA, A₁ receptor activation with N⁶-cyclopentyladenosine (CPA, 50 nM) partially readmitted that effect of VIP (32.6 ± 3.8%). In conclusion, the enhancement of GABA release caused by VIP in hippocampal nerve terminals is dependent on the tonic actions of adenosine on both A₁ and A_2A receptors, and this action of adenosine is essential to VIP modulation of GABA release.     

Activity-dependent bidirectional regulation of GABAA receptor channels by the 5-HT4 receptor-mediated signalling in rat prefrontal cortical pyramidal neurons

Emerging evidence has implicated a potential role for 5-HT₄ receptors in cognition and anxiolysis. One of the main target structures of 5-HT₄ receptors on 'cognitive and emotional' pathways is the prefrontal cortex (PFC). As GABAergic signalling plays a key role in regulating PFC functions, we examined the effect of 5-HT₄ receptors on GABA_A receptor channels in PFC pyramidal neurons. Application of 5-HT₄ receptor agonists produced either an enhancement or a reduction of GABA-evoked currents in PFC neurons, which are both mediated by anchored protein kinase A (PKA). Although PKA phosphorylation of GABA_A receptor β3 or β1 subunits leads to current enhancement or reduction respectively in heterologous expression systems, we found that β3 and β1 subunits are co-expressed in PFC pyramidal neurons. Interestingly, altering PKA activation levels can change the direction of the dual effect, switching enhancement to reduction and vice versa. In addition, increased neuronal activity in PFC slices elevated the PKA activation level, changing the enhancing effect of 5-HT₄ receptors on the amplitude of GABAergic inhibitory postsynaptic currents (IPSCs) to a reduction. These results suggest that 5-HT₄ receptors can modulate GABAergic signalling bidirectionally, depending on the basal PKA activation levels that are determined by neuronal activity. This modulation provides a unique and flexible mechanism for 5-HT₄ receptors to dynamically regulate synaptic transmission and neuronal excitability in the PFC network.     

Purine metabolism in Leishmania donovani amastigotes and promastigotes

Purine metabolism in Leishmania donovani amastigotes was found to be similar to that of promastigotes with the exception of adenosine metabolism. Adenosine kinase activity in amastigotes is approximately 50-fold greater than in promastigotes. Amastigotes deaminate adenosine to inosine through adenosine deaminase, an enzyme not present in promastigotes. Inosine is cleaved to hypoxanthine and phosphoribosylated by hypoxanthine-guanine phosphoribosyltransferase. Promastigotes cleave adenosine to adenine and deaminate adenine to hypoxanthine via adenase, an enzyme not present in amastigotes. Hypoxanthine is phosphoribosylated by hypoxanthine-guanine phosphoribosyltransferase.