VIPergic neuroprotection in epileptogenesis: challenges and opportunities.

In mesial temporal lobe epilepsy (MTLE), seizures typically arise in the hippocampus or other mesial temporal lobe structures. The aetiology of MTLE epileptogenesis in still unknown, yet putative precipitating events such as trauma, complex febrile seizures, status epilepticus, inflammatory insults, or ischemia have been implicated. MTLE is commonly associated to a high degree of hippocampal sclerosis (HS) leading to frequent anti-epileptic drug refractoriness. Thus, the aim of recent therapeutic strategies has shifted from control of symptomatic seizures to putative prevention of epileptogenic processes. Vasoactive intestinal peptide (VIP) acts as a neurotransmitter, neurotrophic or neuroprotective factor in the central nervous system (CNS), also displaying anti-inflammatory and neurogenic actions. In the hippocampus, a brain area implicated in learning and memory, VIP released from basket cells and/or interneuron-selective interneurons controls GABAergic transmission and pyramidal cell activity influencing hippocampal-dependent synaptic plasticity (long-term potentiation and long-term depression) and cognition. VPAC1 receptor activation enhances hippocampal synaptic transmission by fostering disinhibition, while stimulation of VPAC2 receptors favours pyramidal cell excitability. Interestingly, VIP released from interneurons has potent anti-inflammatory actions, participates in the maintenance of the blood-brain barrier integrity, and strengthens neurogenesis. VPAC1 and VPAC2 receptors play differential roles in the regulation of the neuro-immune interactions. In this context, we gathered here the available information concerning the impact of VIP on neurotransmission and neuronal excitability in MTLE-HS and discuss the preventive use of selective VIP receptor ligands to abrogate epileptogenesis in MTLE-HS by controlling synaptic plasticity, neurogenesis and neuronal survival, neuroinflammation, and blood-brain barrier damage.

Activation of Prejunctional P2x2/3 Heterotrimers by ATP Enhances the Cholinergic Tone in Obstructed Human Urinary Bladders.

The objective of this study was to investigate the role of ATP in cholinergic neurotransmission in the urinary bladder of control men and of patients obstructed as a result of benign prostatic hyperplasia (BPH). Human detrusor samples were collected from 41 patients who submitted to transvesical prostatectomy resulting from BPH and 26 male organ donors. The release of [3H]acetylcholine ([3H]ACh) was evoked by electrical field stimulation (10 Hz, 200 pulses) in urothelium-denuded detrusor strips. Myographic recordings were performed to test detrusor strip sensitivity to ACh and ATP. Nerve-evoked [3H]ACh release was 1.5-fold higher in detrusor strips from BPH patients compared with controls. This difference was abolished after desensitization of ionotropic P2X1-3 receptors with an ATP analog, α,ß-methylene ATP (30 µM, applied for 15 minutes). TNP-ATP (10 nM, a preferential P2X2/3 antagonist) and A317491 (100 nM, a selective P2X3 antagonist) were about equipotent in decreasing nerve-evoked [3H]ACh release in control detrusor strips, but the selective P2X1 receptor antagonist NF023 (3 µM) was devoid of effect. The inhibitory effect of TNP-ATP (10 nM) increased from 27% ± 9% to 43% ± 6% in detrusor strips of BPH patients, but the effect of A317491 (100 nM) [3H]ACh release unaltered (20% ± 2% vs. 24% ± 4%). The amplitude of ACh (0.1-100 µM)-induced myographic recordings decreased, whereas sensitivity to ATP (0.01-3 mM) increased in detrusor strips from BPH patients. Besides the well characterized P2X1 receptor-mediated contractile activity of ATP in pathologic human bladders, we show here for the first time that cholinergic hyperactivity in the detrusor of BPH patients is facilitated by activation of ATP-sensitive P2X2/3 heterotrimers. SIGNIFICANCE STATEMENT: Bladder outlet obstruction often leads to detrusor overactivity and reduced bladder compliance in parallel to atropine-resistant increased purinergic tone. Our data show that P2X1 purinoceptors are overexpressed in the detrusor of patients with benign prostatic hyperplasia. Besides the P2X1 receptor-mediated detrusor contractions, ATP favors nerve-evoked acetylcholine release via the activation of prejunctional P2X2/3 excitatory receptors in these patients Thus, our hypothesis is that manipulation of the purinergic tone may be therapeutically useful to counteract cholinergic overstimulation in obstructed patients.

Mesenchymal Stem Cells Ageing: Targeting the "Purinome" to Promote Osteogenic Differentiation and Bone Repair.

Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into bone forming cells. Such ability is compromised in elderly individuals resulting in bone disorders such as osteoporosis, also limiting their clinical usage for cell transplantation and bone tissue engineering strategies. In bone marrow niches, adenine and uracil nucleotides are important local regulators of osteogenic differentiation of MSCs. Nucleotides can be released to the extracellular milieu under both physiological and pathological conditions via (1) membrane cell damage, (2) vesicle exocytosis, (3) ATP-binding cassette transporters, and/or (4) facilitated diffusion through maxi-anion channels, hemichannels or ligand-gated receptor pores. Nucleotides and their derivatives act via adenosine P1 (A1 , A2A , A2B , and A3 ) and nucleotide-sensitive P2 purinoceptors comprising ionotropic P2X and G-protein-coupled P2Y receptors. Purinoceptors activation is terminated by membrane-bound ecto-nucleotidases and other ecto-phosphatases, which rapidly hydrolyse extracellular nucleotides to their respective nucleoside 5'-di- and mono-phosphates, nucleosides and free phosphates, or pyrophosphates. Current knowledge suggests that different players of the "purinome" cascade, namely nucleotide release sites, ecto-nucleotidases and purinoceptors, orchestrate to fine-tuning regulate the activity of MSCs in the bone microenvironment. Increasing studies, using osteoprogenitor cell lines, animal models and, more recently, non-modified MSCs from postmenopausal women, raised the possibility to target chief components of the purinergic signaling pathway to regenerate the ability of aged MSCs to differentiate into functional osteoblasts. This review summarizes the main findings of those studies, prompting for novel therapeutic strategies to control ageing disorders where bone destruction exceeds bone formation, like osteoporosis, rheumatoid arthritis, and fracture mal-union. J. Cell. Physiol. 231: 1852-1861, 2016. © 2016 Wiley Periodicals, Inc.

Impairment of ATP hydrolysis decreases adenosine A1 receptor tonus favoring cholinergic nerve hyperactivity in the obstructed human urinary bladder.

This study was designed to investigate whether reduced adenosine formation linked to deficits in extracellular ATP hydrolysis by NTPDases contributes to detrusor neuromodulatory changes associated with bladder outlet obstruction in men with benign prostatic hyperplasia (BPH). The kinetics of ATP catabolism and adenosine formation as well as the role of P1 receptor agonists on muscle tension and nerve-evoked [(3)H]ACh release were evaluated in mucosal-denuded detrusor strips from BPH patients (n = 31) and control organ donors (n = 23). The neurogenic release of ATP and [(3)H]ACh was higher (P < 0.05) in detrusor strips from BPH patients. The extracellular hydrolysis of ATP and, subsequent, adenosine formation was slower (t (1/2) 73 vs. 36 min, P < 0.05) in BPH detrusor strips. The A(1) receptor-mediated inhibition of evoked [(3)H]ACh release by adenosine (100 µM), NECA (1 µM), and R-PIA (0.3 µM) was enhanced in BPH bladders. Relaxation of detrusor contractions induced by acetylcholine required 30-fold higher concentrations of adenosine. Despite VAChT-positive cholinergic nerves exhibiting higher A(1) immunoreactivity in BPH bladders, the endogenous adenosine tonus revealed by adenosine deaminase is missing. Restoration of A1 inhibition was achieved by favoring (1) ATP hydrolysis with apyrase (2 U mL(-1)) or (2) extracellular adenosine accumulation with dipyridamole or EHNA, as these drugs inhibit adenosine uptake and deamination, respectively. In conclusion, reduced ATP hydrolysis leads to deficient adenosine formation and A(1) receptor-mediated inhibition of cholinergic nerve activity in the obstructed human bladder. Thus, we propose that pharmacological manipulation of endogenous adenosine levels and/or A(1) receptor activation might be useful to control bladder overactivity in BPH patients.

P2X7 receptor antagonist improves gastrointestinal disorders in spontaneously hypertensive rats.

The purinergic system participates in the control of blood pressure. Hypertension promotes the occurrence of gastrointestinal disorders such as intestinal inflammation and gastric emptying delay. This study aimed i) to investigate the participation of the P2X7 receptor blocker Brilliant Blue G (BBG) on gastric emptying of solids and changes in oxidative stress in the gastric fundus, duodenum, and colon of spontaneously hypertensive rats (SHR) and ii) to study the putative relationship of this effect with the renin-angiotensin system. Rats were divided into five groups: Control, SHR, SHR+BBG, SHR+BBG+ATP, and SHR+BBG+ANG II. In the gastrointestinal tract, we assessed gastric emptying (GE) and oxidative stress markers (NOx, MPO, GSH, SOD). We observed a decrease in the GE rate (P<0.05) in SHR vs control rats (21.8±2.0% vs 42.8±3.5%). The decrease in GE was returned (P<0.05) to control levels by BBG in SHR rats (21.8±2.0% vs 41.6±3.2%). Co-administration of ATP or ANG II together with BBG bypassed the effect of the P2X7 antagonist on GE in SHR (P<0.05) (21.9±5.0% vs 25.6±3.0% vs 41.6±3.2%). The MPO activity increased (P<0.05) in the gastric fundus of SHR compared to control rats (6.12±2.26 vs 0.077±0.02 UMPO/mg tissue); this effect was prevented (P<0.05) by BBG (0.55±0.15 vs 6.12±2.26 UMPO/mg tissue). Data demonstrated that blockage of P2X7 receptors with BBG can improve the GE delay and oxidative stress biomarkers in SHR animals. This preventive effect of BBG on GE delay was abrogated by ANG II and ATP, thus prompting crosstalk between renin-angiotensin and the purinergic signaling systems underlying this phenomenon.

Role of ecto-NTPDases on UDP-sensitive P2Y(6) receptor activation during osteogenic differentiation of primary bone marrow stromal cells from postmenopausal women.

This study aimed at investigating the expression and function of uracil nucleotide-sensitive receptors (P2Y(2), P2Y(4), and P2Y(6)) on osteogenic differentiation of human bone marrow stromal cells (BMSCs) in culture. Bone marrow specimens were obtained from postmenopausal female patients (68 ± 5 years old, n = 18) undergoing total hip arthroplasty. UTP and UDP (100 µM) facilitated osteogenic differentiation of the cells measured as increases in alkaline phosphatase (ALP) activity, without affecting cell proliferation. Uracil nucleotides concentration-dependently increased [Ca(2+)](i) in BMSCs; their effects became less evident with time (7 > 21 days) of the cells in culture. Selective activation of P2Y(6) receptors with the stable UDP analog, PSB 0474, mimicked the effects of both UTP and UDP, whereas UTPγS was devoid of effect. Selective blockade of P2Y(6) receptors with MRS 2578 prevented [Ca(2+)](i) rises and osteogenic differentiation caused by UDP at all culture time points. BMSCs are immunoreactive against P2Y(2), P2Y(4), and P2Y(6) receptors. While the expression of P2Y(6) receptors remained fairly constant (7∼21 days), P2Y(2) and P2Y(4) became evident only in less proliferative and more differentiated cultures (7 < 21 days). The rate of extracellular UTP and UDP inactivation was higher in less proliferative and more differentiated cell populations. Immunoreactivity against NTPDase1, -2, and -3 rises as cells differentiate (7 < 21 days). Data show that uracil nucleotides are important regulators of osteogenic cells differentiation predominantly through the activation of UDP-sensitive P2Y(6) receptors coupled to increases in [Ca(2+)](i) . Endogenous actions of uracil nucleotides may be balanced through specific NTPDases determining whether osteoblast progenitors are driven into proliferation or differentiation.

On the role of subtype selective adenosine receptor agonists during proliferation and osteogenic differentiation of human primary bone marrow stromal cells.

Purines are important modulators of bone cell biology. ATP is metabolized into adenosine by human primary osteoblast cells (HPOC); due to very low activity of adenosine deaminase, the nucleoside is the end product of the ecto-nucleotidase cascade. We, therefore, investigated the expression and function of adenosine receptor subtypes (A(1) , A(2A) , A(2B) , and A(3) ) during proliferation and osteogenic differentiation of HPOC. Adenosine A(1) (CPA), A(2A) (CGS21680C), A(2B) (NECA), and A(3) (2-Cl-IB-MECA) receptor agonists concentration-dependently increased HPOC proliferation. Agonist-induced HPOC proliferation was prevented by their selective antagonists, DPCPX, SCH442416, PSB603, and MRS1191. CPA and NECA facilitated osteogenic differentiation measured by increases in alkaline phosphatase (ALP) activity. This contrasts with the effect of CGS21680C which delayed HPOC differentiation; 2-Cl-IB-MECA was devoid of effect. Blockade of the A(2B) receptor with PSB603 prevented osteogenic differentiation by NECA. In the presence of the A(1) antagonist, DPCPX, CPA reduced ALP activity at 21 and 28 days in culture. At the same time points, blockade of A(2A) receptors with SCH442416 transformed the inhibitory effect of CGS21680C into facilitation. Inhibition of adenosine uptake with dipyridamole caused a net increase in osteogenic differentiation. The presence of all subtypes of adenosine receptors on HPOC was confirmed by immunocytochemistry. Data show that adenosine is an important regulator of osteogenic cell differentiation through the activation of subtype-specific receptors. The most abundant A(2B) receptor seems to have a consistent role in cell differentiation, which may be balanced through the relative strengths of A(1) or A(2A) receptors determining whether osteoblasts are driven into proliferation or differentiation.

Tetanic failure due to decreased endogenous adenosine A(2A) tonus operating neuronal Ca(v) 1 (L-type) influx in Myasthenia gravis.

In healthy motor endplates, tetanic depression is overcome by tonic adenosine A(2A) -receptor-mediated facilitation of transmitter release. The A(2A) receptor operates a coordinated shift from fast-desensitizing Ca(v) 2.1 (P/Q) calcium influx to long-lasting Ca(V) 1 (L) channels on motor nerve terminals. This study aimed at investigating whether A(2A) receptors-operated Ca(2+) influx via Ca(V) 1 (L)-type channels contribute to sustain acetylcholine release evoked by 50 Hz-bursts in toxin-induced Myasthenia gravis (TIMG) rats. In contrast to control animals, inhibition of [(3) H]acetylcholine (ACh) release by the Ca(V) 2.1 (P/Q) channel blocker, ω-Agatoxin IVA (100 nM), in TIMG rats had a higher magnitude than that observed with the Ca(V) 1 (L) channel blocker, nifedipine (1 µM). Adenosine deaminase (0.5 U/mL) and the A(2A) receptor antagonist, ZM 241385 (50 nM), decreased [(3) H]ACh release by a similar amount in control rats, but their effects were smaller in magnitude in myasthenic animals. The adenosine precursor, AMP (100 µM), increased (~40%) ACh release in both control and TIMG animals. Blockade of A(2A) , but not of A(1) , receptors prevented AMP-induced facilitation of transmitter release; nifedipine (1 µM) mimicked the effect of the A(2A) receptor antagonist. Video-microscopy studies designed to measure real-time transmitter exocytosis using the FM4-64 fluorescent dye fully supported radiochemical data. Thus, impairment of the adaptive shift from Ca(V) 2.1 (P/Q) to Ca(V) 1 (L) channels may contribute to tetanic failure in myasthenic rats. This parallels the reduction of adenosine A(2A) receptor tonus in TIMG animals, which might be restored by exogenous application of AMP.

Presynaptic muscarinic and adenosine receptors are involved in 2 Hz-induced train-of-four fade caused by antinicotinic neuromuscular relaxants in the rat.

1. Train-of-four fade (TOF(fade) ) is a clinically useful parameter to monitor the degree of block of neuromuscular transmission in curarized patients. Experimentally, TOF(fade) has been attributed to the blockade of facilitatory nicotinic receptors on motor nerve terminals. There is less information regarding the involvement of coexistent presynaptic receptors (e.g. muscarinic M(1) and M(2) , adenosine A(1) and A(2A) ) in the TOF(fade) produced by antinicotinic agents. 2. In the present study, we evaluated the TOF(fade) caused by antinicotinic neuromuscular relaxants (hexamethonium, d-tubocurarine, vecuronium and rocuronium) as the ratio of the muscle tension produced in the rat diaphragm by the fourth to the first stimulus (T(4) /T(1) ) of a train-of-four stimuli delivered to the phrenic nerve trunk at a frequency of 2 Hz. 3. All antinicotinic agents, except hexamethonium, decreased the amplitude of muscle tension during the first stimulus. Hexamethonium, (5.47 mmol/L), d-tubocurarine- (1.1 µmol/L), vecuronium (4.7 µmol/L)- and rocuronium (9.8 µmol/L)-induced TOF(fade) was attenuated by 10 nmol/L pirenzepine (an M(1) receptor antagonist), 1 µmol/L methoctramine (an M(2) receptor antagonist) and 2.5 nmol/L 1,3-dipropyl-8-cyclopentylxanthine (an A(1) receptor antagonist). Blockade of the A(2A) receptor with 10 nmol/L ZM241385 partially reversed the TOF(fade) induced by d-tubocurarine, vecuronium and rocuronium, but not that caused by the 'pure' neuronal nicotinic receptor antagonist hexamethonium, unless one increased the concentration of ZM241385 to 50 nmol/L. 4. The data indicate that presynaptic M(1) , M(2) , A(1) and A(2A) receptors play a role in neuromuscular TOF(fade) caused by antinicotinic neuromuscular relaxants. Such interplay depends on adenosine tonus and on the affinity of neuromuscular blocking agents for neuronal versus muscular nicotinic receptors.

Tuning adenosine A1 and A2A receptors activation mediates L-citrulline-induced inhibition of [3H]-acetylcholine release depending on nerve stimulation pattern.

The influence of nerve stimulation pattern on transmitter release inhibition by L-citrulline, the co-product of NO biosynthesis by nitric oxide synthase (NOS), was studied in the rat phrenic nerve-hemidiaphragm. We also investigated the putative interactions between NOS pathway and the adenosine system. L-citrulline (10-470 microM), the NOS substrate L-arginine (10-470 microM) and the NO donor 3-morpholinylsydnoneimine (SIN-1, 1-10 microM), concentration-dependently inhibited [(3)H]-acetylcholine ([(3)H]-ACh) release from rat motor nerve endings. Increasing stimulus frequency from 5 Hz-trains to 50 Hz-bursts enhanced [(3)H]-ACh release inhibition by l-arginine (47 microM) and L-citrulline (470 microM), whereas the effect of SIN-1 (10 microM) remained unchanged. NOS inhibition with N(omega)-nitro-L-arginine (100 microM) prevented the effect of L-arginine, but not that of L-citrulline. Adenosine deaminase (2.5 U/ml) and the adenosine transport inhibitor, S-(p-nitrobenzyl)-6-thioinosine (10 microM), attenuated release inhibition by L-arginine and L-citrulline. With 5 Hz-trains, blockade of A(1) receptors with 1,3-dipropyl-8-cyclopentyl xanthine (2.5 nM), but not of A(2A) receptors with ZM241385 (10nM), reduced the inhibitory action of l-arginine and L-citrulline; the opposite was verified with 50 Hz-bursts. Blockade of muscarinic M(2) autoreceptors with AF-DX116 (10 nM) also attenuated the effects of L-arginine and L-citrulline with 50 Hz-bursts. L-citrulline (470 microM) increased basal adenosine outflow via the equilibrative nucleoside transport system sensitive to NBTI (10 microM), without significantly (P>0.05) changing the nucleoside release subsequent to nerve stimulation. Data indicate that NOS-derived L-citrulline negatively modulates [(3)H]-ACh release by increasing adenosine outflow channelling to A(1) and A(2A) receptors activation depending on the stimulus paradigm. While adenosine acts predominantly at inhibitory A(1) receptors during 5 Hz-trains, inhibition of ACh release by L-citrulline at 50 Hz-bursts depends on the interplay between adenosine A(2A) and muscarinic M(2) receptors.

P2X7 receptor antagonist improves gastrointestinal disorders in spontaneously hypertensive rats

The purinergic system participates in the control of blood pressure. Hypertension promotes the occurrence of gastrointestinal disorders such as intestinal inflammation and gastric emptying delay. This study aimed i) to investigate the participation of the P2X7 receptor blocker Brilliant Blue G (BBG) on gastric emptying of solids and changes in oxidative stress in the gastric fundus, duodenum, and colon of spontaneously hypertensive rats (SHR) and ii) to study the putative relationship of this effect with the renin-angiotensin system. Rats were divided into five groups: Control, SHR, SHR+BBG, SHR+BBG+ATP, and SHR+BBG+ANG II. In the gastrointestinal tract, we assessed gastric emptying (GE) and oxidative stress markers (NOx, MPO, GSH, SOD). We observed a decrease in the GE rate (P<0.05) in SHR vs control rats (21.8±2.0% vs 42.8±3.5%). The decrease in GE was returned (P<0.05) to control levels by BBG in SHR rats (21.8±2.0% vs 41.6±3.2%). Co-administration of ATP or ANG II together with BBG bypassed the effect of the P2X7 antagonist on GE in SHR (P<0.05) (21.9±5.0% vs 25.6±3.0% vs 41.6±3.2%). The MPO activity increased (P<0.05) in the gastric fundus of SHR compared to control rats (6.12±2.26 vs 0.077±0.02 UMPO/mg tissue); this effect was prevented (P<0.05) by BBG (0.55±0.15 vs 6.12±2.26 UMPO/mg tissue). Data demonstrated that blockage of P2X7 receptors with BBG can improve the GE delay and oxidative stress biomarkers in SHR animals. This preventive effect of BBG on GE delay was abrogated by ANG II and ATP, thus prompting crosstalk between renin-angiotensin and the purinergic signaling systems underlying this phenomenon.

L-citrulline inhibits [3H]acetylcholine release from rat motor nerve terminals by increasing adenosine outflow and activation of A1 receptors.

BACKGROUND AND PURPOSE: Nitric oxide (NO) production and depression of neuromuscular transmission are closely related, but little is known about the role of L-citrulline, a co-product of NO biosynthesis, on neurotransmitter release. EXPERIMENTAL APPROACH: Muscle tension recordings and outflow experiments were performed on rat phrenic nerve-hemidiaphragm preparations stimulated electrically. KEY RESULTS: L-citrulline concentration-dependently inhibited evoked [(3)H]ACh release from motor nerve terminals and depressed nerve-evoked muscle contractions. The NO synthase (NOS) substrate, L-arginine, and the NO donor, 3-morpholinosydnonimine chloride (SIN-1), also inhibited [(3)H]ACh release with a potency order of SIN-1>L-arginine>L-citrulline. Co-application of L-citrulline and SIN-1 caused additive effects. NOS inactivation with N(omega)-nitro-L-arginine prevented L-arginine inhibition, but not that of L-citrulline. The NO scavenger, haemoglobin, abolished inhibition of [(3)H]ACh release caused by SIN-1, but not that caused by L-arginine. Inactivation of guanylyl cyclase with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) fully blocked SIN-1 inhibition, but only partially attenuated the effects of L-arginine. Reduction of extracellular adenosine accumulation with adenosine deaminase or with the nucleoside transport inhibitor, S-(p-nitrobenzyl)-6-thioinosine, attenuated the effects of L-arginine and L-citrulline, while not affecting inhibition by SIN-1. Similar results were obtained with the selective adenosine A(1) receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine. L-citrulline increased the resting extracellular concentration of adenosine, without changing that of the adenine nucleotides. CONCLUSIONS AND IMPLICATIONS: NOS catalyses the formation of two neuronally active products, NO and L-citrulline. While, NO may directly reduce transmitter release through stimulation of soluble guanylyl cyclase, the inhibitory action of L-citrulline may be indirect through increasing adenosine outflow and subsequently activating inhibitory A(1) receptors.

Inverse gradient of nitrergic and purinergic inhibitory cotransmission in the mouse colon.

AIM: Gastrointestinal smooth muscle relaxation is accomplished by the neural corelease of ATP or a related purine and nitric oxide. Contractions are triggered by acetylcholine and tachykinins. The aim of this work was to study whether regional differences in neurotransmission could partially explain the varied physiological roles of each colonic area. METHODS: We used electrophysiological and myography techniques to evaluate purinergic (L-NNA 1 mm incubated tissue), nitrergic (MRS2500 0.3 µm incubated tissue) and cholinergic neurotransmission (L-NNA 1 mm and MRS2500 0.3 µm incubated tissue) in the proximal, mid and distal colon of CD1 mice (n = 42). RESULTS: Purinergic electrophysiological responses elicited by single pulses (28 V) were greater in the distal (IJPfMAX = -35.3 ± 2.2 mV), followed by the mid (IJPfMAX = -30.6 ± 1.0 mV) and proximal (IJPfMAX = -11.7 ± 1.1 mV) colon. In contrast, nitrergic responses decreased from the proximal colon (IJPsMAX = -11.4 ± 1.1 mV) to the mid (IJPsMAX = -9.1 ± 0.4 mV), followed by the distal colon (IJPsMAX = -1.8 ± 0.3 mV). A similar rank of order was observed in neural mediated inhibitory mechanical responses including electrical field stimulation-mediated responses and neural tone. ADPßs concentration-response curve was shifted to the left in the distal colon. In contrast, NaNP responses did not differ between regions. Cholinergic neurotransmission elicited contractions of a similar amplitude throughout the colon. CONCLUSION: An inverse gradient of purinergic and nitrergic neurotransmission exists through the mouse colon. The proximal and mid colon have a predominant nitrergic neurotransmission probably due to the fact that their storage function requires sustained relaxations. The distal colon, in contrast, has mainly purinergic neurotransmission responsible for the phasic relaxations needed to propel dehydrated faeces.

P2X7 receptor activation downmodulates Na(+)-dependent high-affinity GABA and glutamate transport into rat brain cortex synaptosomes.

Sodium-dependent high-affinity amino-acid transporters play crucial roles in terminating synaptic transmission in the central nervous system (CNS). However, there is lack of information about the mechanisms underlying the regulation of amino-acid transport by fast-acting neuromodulators, like ATP. Here, we investigated whether activation of the ATP-sensitive P2X7 receptor modulates Na(+)-dependent high-affinity γ-aminobutyric acid (GABA) and glutamate uptake into nerve terminals (synaptosomes) of the rat cerebral cortex. Radiolabeled neurotransmitter accumulation was evaluated by liquid scintillation spectrometry. The cell-permeant sodium-selective fluorescent indicator, SBFI-AM, was used to estimate Na(+) influx across plasma membrane. 2'(3')-O-(4-benzoylbenzoyl)ATP (BzATP, 3-300 µM), a prototypic P2X7 receptor agonist, concentration-dependently decreased [(3)H]GABA (14%) and [(14)C]glutamate (24%) uptake; BzATP decreased transport maximum velocity (Vmax) without affecting the Michaelis constant (Km) values. The selective P2X7 receptor antagonist, A-438079 (3 µM), prevented inhibition of [(3)H]GABA and [(14)C]glutamate uptake by BzATP (100 µM). The inhibitory effect of BzATP coincided with its ability to increase intracellular Na(+) and was mimicked by Na(+) ionophores, like gramicidin and monensin. Increases in intracellular Na(+) (with veratridine or ouabain) or substitution of extracellular Na(+) by N-methyl-D-glucamine (NMDG)(+) all decreased [(3)H]GABA and [(14)C]glutamate uptake and attenuated BzATP effects. Uptake inhibition by BzATP (100 µM) was also attenuated by calmidazolium, which selectively inhibits Na(+) currents through the P2X7 receptor pore. In conclusion, disruption of the Na(+) gradient by P2X7 receptor activation downmodulates high-affinity GABA and glutamate uptake into rat cortical synaptosomes. Interference with amino-acid transport efficacy may constitute a novel target for therapeutic management of cortical excitability.

Amplification of neuromuscular transmission by methylprednisolone involves activation of presynaptic facilitatory adenosine A2A receptors and redistribution of synaptic vesicles.

The mechanisms underlying improvement of neuromuscular transmission deficits by glucocorticoids are still a matter of debate despite these compounds have been used for decades in the treatment of autoimmune myasthenic syndromes. Besides their immunosuppressive action, corticosteroids may directly facilitate transmitter release during high-frequency motor nerve activity. This effect coincides with the predominant adenosine A2A receptor tonus, which coordinates the interplay with other receptors (e.g. muscarinic) on motor nerve endings to sustain acetylcholine (ACh) release that is required to overcome tetanic neuromuscular depression in myasthenics. Using myographic recordings, measurements of evoked [(3)H]ACh release and real-time video microscopy with the FM4-64 fluorescent dye, results show that tonic activation of facilitatory A2A receptors by endogenous adenosine accumulated during 50 Hz bursts delivered to the rat phrenic nerve is essential for methylprednisolone (0.3 mM)-induced transmitter release facilitation, because its effect was prevented by the A2A receptor antagonist, ZM 241385 (10 nM). Concurrent activation of the positive feedback loop operated by pirenzepine-sensitive muscarinic M1 autoreceptors may also play a role, whereas the corticosteroid action is restrained by the activation of co-expressed inhibitory M2 and A1 receptors blocked by methoctramine (0.1 µM) and DPCPX (2.5 nM), respectively. Inhibition of FM4-64 loading (endocytosis) by methylprednisolone following a brief tetanic stimulus (50 Hz for 5 s) suggests that it may negatively modulate synaptic vesicle turnover, thus increasing the release probability of newly recycled vesicles. Interestingly, bulk endocytosis was rehabilitated when methylprednisolone was co-applied with ZM241385. Data suggest that amplification of neuromuscular transmission by methylprednisolone may involve activation of presynaptic facilitatory adenosine A2A receptors by endogenous adenosine leading to synaptic vesicle redistribution.

Adenosine uptake and deamination regulate tonic A2a receptor facilitation of evoked [3H]acetylcholine release from the rat motor nerve terminals.

The actions of adenosine, adenosine deaminase, the adenosine uptake blocker, S-(p-nitrobenzyl)-6-thioinosine, and of the adenosine deaminase inhibitor, erythro-9(2-hydroxy-3-nonyl)adenine, on electrically evoked [3H]acetylcholine release were investigated in rat phrenic nerve-hemidiaphragm preparations. Adenosine deaminase (0.25-2.5 U/ml) increased [3H]acetylcholine release. S-(p-Nitrobenzyl)-6-thioinosine (3-30 microM) and erythro-9(2-hydroxy-3-nonyl)adenine (25 nM-50 microM) caused biphasic effects on [3H]acetylcholine release: at low concentrations S-(p-nitrobenzyl)-6-thiomosine (5 microM) and erythro-9(2-hydroxy-3-nonyl)adeNine (50 nM) decreased [3H]acetylcholine release, and at concentrations higher than 10 microM S-(p-nitrobenzyl)-6-thioinosine and 0.5 microM for erythro-9(2-hydroxy-3-nonyl)adenine facilitated [3H]acetylcholine release. Both S-(p-nitrobenzyl)-6-thioinosine-induced inhibition and facilitation of [3H]acetylcholine release resulted from extracellular endogenous adenosine accumulation, because they were blocked after inactivation of endogenous adenosine with adenosine deaminase (0.5 U/ml). The inhibitory actions of both S-(p-nitrobenzyl)-6-thioinosine (5 microM) and erythro-9(2-hydroxy-3-nonyl)adenine (50 nM) were antagonized by the A1 adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (2.5 nM), whereas the blockade of A2a adenosine receptors with PD 115,199 (25 nM) prevented the facilitatory effects of S-(p-nitrobenzyl)-6-thioinosine (30 microM) and erythro-9(2-hydroxy-3-nonyl)adenine (50 microM). The adenosine deaminase inhibitor, erythro-9(2-hydroxy-3-nonyl)adenine (25 nM), potentiated the effect of S-(p-nitrobenzyl)-6-thioinosine (3-30 microM), and this adenosine uptake blocker, when applied at a concentration (3 microM) that by itself was devoid of effect, potentiated both the inhibitory (25 nM) and excitatory (0.5 microM) effects of erythro-9(2-hydroxy-3-nonyl)adenine, on evoked [3H]acetylcholine release. Exogenously applied adenosine (10-500 microM) had biphasic effects similar to those of S-(p-nitrobenzyl)-6-thioinosine and erythro-9(2-hydroxy-3-nonyl)adenine. Adenosine (30 microM) reduction of evoked [3H]acetylcholine release was prevented after pretreatment with 1,3-dipropyl-8-cyclopentylxanthine (2.5 nM); when applied at high concentrations (100-500 microM), adenosine consistently increased evoked [3H]acetylcholine release in a PD 115,199 (25 nM)-sensitive manner. It is concluded that both uptake and deamination are effective in removing extracellular endogenous adenosine that tonically activates both inhibitory (A1) and excitatory (A2a) adenosine receptors, regulating the A1/A2a adenosine receptors' activation balance.

Potentiation by tonic A2a-adenosine receptor activation of CGRP-facilitated [3H]-ACh release from rat motor nerve endings.

1. The effect of calcitonin gene-related peptide (CGRP) on [3H]-acetylcholine ([3H]-ACh) release from motor nerve endings and its interaction with presynaptic facilitatory A2a-adenosine and nicotinic acetylcholine receptors was studied on rat phrenic nerve-hemidiaphragm preparations loaded with [3H]-choline. 2. CGRP (100-400 nM) increased electrically evoked [3H]-ACh release from phrenic nerve endings in a concentration-dependent manner. 3. The magnitude of CGRP excitation increased with the increase of the stimulation pulse duration from 40 microseconds to 1 ms, keeping the frequency, the amplitude and the train length constants. With 1 ms pulses, the evoked [3H]-ACh release was more intense than with 40 microseconds pulse duration. 4. Both the nicotinic acetylcholine receptor agonist, 1,1-dimethyl-4-phenylpiperazinium, and the A2a adenosine receptor agonist, CGS 21680C, increased evoked [3H]-ACh release, but only CGS 21680C potentiated the facilitatory effect of CGRP. This potentiation was prevented by the A2a adenosine receptor antagonist, PD 115,199. 5. Adenosine deaminase prevented the excitatory effect of CGRP (400 nM) on [3H]-ACh release. This effect was reversed by the non-hydrolysable A2a-adenosine receptor agonist, CGS 21680C. 6. The nicotinic antagonist, tubocurarine, did not significantly change, whereas the A2-adenosine receptor antagonist, PD 115,199, blocked the CGRP facilitation. The A1-adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine, potentiated the CGRP excitatory effect. 7. The results suggest that the facilitatory effect of CGRP on evoked [3H]-ACh release from rat phrenic motor nerve endings depends on the presence of endogenous adenosine which tonically activates A2a-adenosine receptors. Since both CGRP and A2a-adenosine receptors are positively coupled to the adenylate cyclase/cyclic AMP system, cooperation between these receptors might occur at the second messenger transduction system level.

Inhibitory and excitatory effects of adenosine receptor agonists on evoked transmitter release from phrenic nerve ending of the rat.

1. The effects of the adenosine analogues, 5'-N-ethyl-carboxamide adenosine (NECA), R-N6-phenylisopropyladenosine (R-PIA), 2-chloroadenosine (CADO), and CGS 21680C on electrically evoked tritium outflow from preparations loaded with [3H]-choline and on evoked endplate potentials (e.p.ps), as well as the ability of the xanthines, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) and PD 115,199 to antagonize the effects of the adenosine analogues, were investigated in phrenic nerve-diaphragm preparations. 2. NECA, R-PIA and CADO decreased, in a concentration-dependent manner, the evoked tritium outflow from preparations loaded with [3H]-choline. NECA and R-PIA were about equipotent and more potent than CADO. 3. DPCPX shifted to the right in a near parallel fashion the concentration-response curve for the inhibitory effect of R-PIA on evoked tritium outflow. 4. In the presence of DPCPX, NECA increased, rather than decreased, evoked tritium outflow. PD 115,119 antagonized, in a concentration-dependent manner, this excitatory effect of NECA. 5. CGS 21680C, in low nanomolar concentrations, increased evoked tritium outflow, an effect also antagonized by PD 115,119. 6. CGS 21680C increased, and R-PIA decreased, the amplitude of e.p.ps recorded from preparations paralysed with tubocurarine. Both effects could be observed in the same endplate. 7. It is concluded that both inhibitory (probably A1) and excitatory (probably A2) adenosine receptors coexist at the rat neuromuscular junction, modulating the evoked release of acetylcholine.

Preferential activation of excitatory adenosine receptors at rat hippocampal and neuromuscular synapses by adenosine formed from released adenine nucleotides.

1. In the present work, we investigated the action of adenosine originating from extracellular catabolism of adenine nucleotides, in two preparations where synaptic transmission is modulated by both inhibitory A1 and excitatory A(2a)-adenosine receptors, the rat hippocampal Schaffer fibres/CA1 pyramid synapses and the rat innervated hemidiaphragm. 2. Endogenous adenosine tonically inhibited synaptic transmission, since 0.5-2 u ml-1 of adenosine deaminase increased both the population spike amplitude (30 +/- 4%) and field excitatory post-synaptic potential (f.e.p.s.p.) slope (27 +/- 4%) recorded from hippocampal slices and the evoked [3H]-acetylcholine ([3H]-ACh) release from the motor nerve terminals (25 +/- 2%). 3. alpha, beta-Methylene adenosine diphosphate (AOPCP) in concentrations (100-200 microM) that almost completely inhibited the formation of adenosine from the extracellular catabolism of AMP, decreased population spike amplitude by 39 +/- 5% and f.e.p.s.p. slope by 32 +/- 3% in hippocampal slices and [3H]-ACh release from motor nerve terminals by 27 +/- 3%. 4. Addition of exogenous 5'-nucleotidase (5 u ml-1) prevented the inhibitory effect of AOPCP on population spike amplitude and f.e.p.s.p. slope by 43-57%, whereas the P2 antagonist, suramin (100 microM), did not modify the effect of AOPCP. 5. In both preparations, the effect of AOPCP resulted from prevention of adenosine formation since it was no longer evident when accumulation of extracellular adenosine was hindered by adenosine deaminase (0.5-2 u ml-1). The inhibitory effect of AOPCP was still evident when A1 receptors were blocked by 1,3-dipropyl-8-cyclopentylxanthine (2.5-5 nM), but was abolished by the A2 antagonist, 3,7-dimethyl-1-propargylxanthine (10 microM). 6. These results suggest that adenosine originating from catabolism of released adenine nucleotides preferentially activates excitatory A2 receptors in hippocampal CAI pyramid synapses and in phrenic motor nerve endings.

Purinergic regulation of acetylcholine release.

At the neuromuscular junction and possibly also at the synaptic level in the brain, the main sequence of events (see Fig. 5) that involves purines in modulation of ACh release includes the following observations: (1) storage of ATP and its release either together with, or independently of acetylcholine. ATP is also released from the post-junctional component. Adenosine as such is released either from the motor nerve terminals or from the post-junctional component. (2) There is extracellular hydrolysis of ATP to adenosine, which is the active substance to modulate transmitter release. The key enzyme in the conversion of AMP into adenosine is the ecto 5'-nucleotidase. When ecto-5'-nucleotidase is not available (e.g. in cholinergic nerve terminals of the cerebral cortex) ATP as such exerts the neuromodulatory role normally fulfilled by adenosine. (3) Both the inhibition and the excitation induced by adenosine on ACh release in the rat is inactivated through up-take and deamination. (4) Adenosine-induced inhibition of ACh release is mediated via A1 receptors and the excitation via A2a receptors. The A2a receptors are positively coupled to the adenylate cyclase/cyclic AMP system, whereas the presynaptic A1 receptors (a) may be negatively linked to adenylate cyclase and (b) to phospholipase C, and, upon stimulation, (c) increase potassium conductance and (d) decrease calcium conductance. (5) Activation of A2a receptors is essential for substances that facilitate ACh release (e.g. CGRP, forskolin) to exert their effects, as well as for induction of nicotinic autofacilitatory receptor desensitization. (6) There are interactions between A1 and A2a receptors. Thus, the net adenosine neuromodulatory response is the resultant, at each moment, of the relative degree of activation of each one of these receptors. This relative activation depends upon the intensity (frequency, pulse duration) of stimulation of the motor nerve terminals. (7) Adenosine released as such seems to preferentially activate A1 receptors, whereas the adenosine formed from metabolism of adenine nucleotides prefers to activate the A2a receptors. In conclusion, to find out precisely what occurs with ACh in transmitting its message at the synaptic level, one has to consider the subtle ways used by purines to modulate the ACh response. It therefore appears of interest that pharmacological and therapeutic strategies use this knowledge to approach cholinergic transmission deficiencies based upon reduction of ACh release.