Differential effects of nicotinic acetylcholine receptor stimulation on negative occasion setting.

We have previously shown that nicotine enhances learning in a negative occasion setting task in which rats are trained to distinguish between two different trial types. During reinforced trials, a target stimulus (a tone) is presented and immediately followed by food reward. On nonreinforced trials, a feature stimulus (a light) is presented prior to the tone and indicates the absence of reward following presentation of the tone. The goal of the present study was to identify the behavioral mechanism through which nicotine affects this form of learning, and to determine which subtype(s) of nicotinic acetylcholine receptors mediate the effects of nicotine. Consistent with our prior findings, nicotine administration enhanced the ability of rats to discriminate between the two trial types. Nicotine enhanced the magnitude of the discrimination by decreasing responding to the tone on nonreinforced trials. Nicotine-treated rats also learned the discrimination in fewer sessions than control rats. A significant new finding was that nicotine also increased the orienting response to the light, suggesting that nicotine may enhance learning the serial feature negative discrimination by increasing attention to the visual feature. In addition, we found that RJR-2403, a selective α4β2 nicotinic receptor agonist, also enhanced discrimination. However, RJR-2403 did not affect responding on nonreinforced trials, nor did RJR-2403 affect orienting to the light. Together these data indicate that nicotine may enhance discrimination by enhancing tone-reward associability through α4β2 nicotinic receptors and by enhancing attention to the light through non-α4β2 receptor subtypes. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Adenosine A1 and A2 receptor agonists significantly prevent the electroencephalographic effects induced by MK-801 in rats

Both N6-cyclopentyladenosine (CPA, adenosine A1 receptor agonist) and 2-[4-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamido-adenosi ne (CGS 21680, adenosine A2 receptor agonist) inhibited the electroencephalographic (EEG) effects induced by the noncompetitive NMDA receptor antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo-(a,d)cyclohepten-5,10-imine maleate (MK-801) in rats. While the inhibitory effects of CPA were evident at doses (0.1 and 0.5 mg/kg i.p.) devoid of intrinsic behavioral effects, CGS 21680 was effective only when administered at depressant doses (2 mg/kg i.p.). Since the effects induced by NMDA receptor antagonists may be regarded as a model of psychosis, these results suggest a possible role of adenosine receptor agonists as antipsychotics.     

Adenosine receptor mediated stimulation of intracellular calcium in acutely isolated astrocytes

The characteristics of adenosine receptors found in glial fibrillary acid protein (GFAP)-positive astrocytes acutely isolated from the cerebral cortices of 4- to 12-day old rats were examined by evaluating the effects of adenosine and its analogues on intracellular calcium levels. First, these effects were compared with those seen in primary astrocytic cultures, and it was found that acutely isolated astrocytes showed much greater sensitivity to adenosine than their cultured counterparts. Then, the adenosine evoked calcium responses in acutely isolated cells were evaluated under various conditions. The responses to adenosine were not inhibited by papaverine, an uptake blocker, or by removal of extracellular calcium. U73122, a phospholipase C inhibitor, was able to completely inhibit the adenosine response. The receptor inhibitor 3-isobutyl-1-methylxanthine inhibited the calcium response to adenosine, providing evidence that the response is not coupled to the xanthine-insensitive A3 receptor. The stimulatory action of NECA, a non-selective analogue, was blocked neither by the A2A-selective receptor antagonist 8-(3-chlorostyryl) caffeine nor by the A1-selective receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. The A2B receptor antagonist alloxazine, however, was able to completely inhibit the increase in intracellular calcium produced by NECA. Taken together, these data suggest that the adenosine-evoked calcium response in acutely isolated astrocytes is coupled to the A2B receptor.     

Positive chronotropic and negative inotropic effects induced by potassium chloride in the isolated canine atrium

The effects of potassium chloride on inotropic and chronotropic activity were investigated in five isolated canine atrium preparations which were suspended in a bath and perfused with arterial blood from the carotid artery of the heparinized support dog. Potassium chloride administered into the cannulated sinus node artery in a dose range of 100 mug-1 mg produced a dose-related negative inotropic and a positive chronotropic effect. These effects were not influenced by treatment with either atropine or propranolol. From these results, it is concluded that potassium had a direct negative effect on atrial contractility and a direct positive effect on atrial rate.     

Adenosine deaminase and A1 adenosine receptors internalize together following agonist-induced receptor desensitization

A1 adenosine receptors (A1Rs) and adenosine deaminase (ADA; EC 3.5.4. 4) interact on the cell surface of DDT1MF-2 smooth muscle cells. The interaction facilitates ligand binding and signaling via A1R, but it is not known whether it has a role in homologous desensitization of A1Rs. Here we show that chronic exposure of DDT1MF-2 cells to the A1R agonist, N6-(R)-(phenylisopropyl)adenosine (R-PIA), caused a rapid aggregation or clustering of A1 receptor molecules on the cell membrane, which was enhanced by pretreatment with ADA. Colocalization between A1R and ADA occurred in the R-PIA-induced clusters. Interestingly, colocalization between A1R and ADA also occurred in intracellular vesicles after internalization of both protein molecules in response to R-PIA. Agonist-induced aggregation of A1Rs was mediated by phosphorylation of A1Rs, which was enhanced and accelerated in the presence of ADA. Ligand-induced second-messenger desensitization of A1Rs was also accelerated in the presence of exogenous ADA, and it correlated well with receptor phosphorylation. However, although phosphorylation of A1R returned to its basal state within minutes, desensitization continued for hours. The loss of cell-surface binding sites (sequestration) induced by the agonist was time-dependent (t1/2= 10 +/- 1 h) and was accelerated by ADA. All of these results strongly suggest that ADA plays a key role in the regulation of A1Rs by accelerating ligand-induced desensitization and internalization and provide evidence that the two cell surface proteins internalize via the same endocytic pathway.     

The PKC activator PMA preconditions rabbit heart in the presence of adenosine receptor blockade: is 5'-nucleotidase important?

While there is good evidence that both protein kinase C (PKC) and adenosine are involved in ischemic preconditioning, their sequence in the intracellular signaling cascade is in dispute. One hypothesis proposes that PKC activation causes release of adenosine which then protects the heart, while the other proposes that adenosine stimulates PKC which in turn causes protection. Accordingly, we studied the effects of specified sequences of pharmacologic triggers and blockers on the infarct-sparing effect of a preconditioning protocol. The combination of the adenosine receptor agonist R(-)N6-(2-phenylisopropyl) adenosine (PIA) and the PKC blocker chelerythrine would be protective only if the first hypothesis were correct. On the other hand, the combination of the adenosine receptor blocker 8-(p-sulfophenyl) theophylline (SPT) and a PKC activator would be protective only if the second hypothesis were correct. Isolated, Krebs-perfused rabbit hearts experienced 30 min of regional ischemia and 2 h of reperfusion. Infarct size was quantitated by triphenyltetrazolium chloride staining. In untreated control hearts, 30.0 +/- 2.7% of the risk zone infarcted. Fifty nmol/l PIA for 20 min starting 10 min prior to ischemia resulted in only 8.4 +/- 1.9% infarction (P<0.01), while the combination of PIA and 5 micromol/l chelerythrine resulted in large infarcts of 27.8 +/- 3.2%. This attenuation of the protective effect continued to be observed even when the PIA infusion was continued to the end of the reperfusion period. Conversely, 0.2 nmol of the PKC activator phorbol 12-myristate 13-acetate (PMA) infused during the 10-min interval prior to ischemia protected the hearts (6.5 +/- 1.3% infarction, P<0.01 v control). And protection persisted when PMA-treated hearts were also exposed to 100 microM SPT for 35 min starting 5 min prior to ischemia (9.5 +/- 1.9% infarction, P<0.01 v control). When PKC activation by the PKC-coupled agonist phenylephrine was continued to the end of ischemia and adenosine blockade was extended throughout the reperfusion period by prolonged infusion of SPT, protection was unaffected. The administration of either SPT or chelerythrine alone did not confer any protection (32.5 +/- 3.3 and 34.0 +/- 3.2% infarction, respectively). Thus, because the combination of PKC activation and adenosine receptor blockade was protective while that of adenosine receptor agonist and PKC blockade was not, adenosine receptors must be upstream of PKC in preconditioning.     

The role of calcium in the negative inotropic effect of lanthanum

Lanthanum reduces the rate of calcium fast exchanging processes, whereas the slowly exchanging phase is not modified. Experimental data also confirm the importance of calcium fast exchanging fraction in regulating contractile force of cardiac muscle.     

Adenosine A1 receptor agonist GR79236 suppresses apnea during all sleep stages in the rat

We tested the hypothesis that N-[(1S, trans)-2-hydroxycyclopentyl]adenosine (GR79236), a novel adenosine A1 receptor agonist, would suppress sleep-related apnea in the rat at doses not associated with hypotension or hypothermia. Nine adult Sprague-Dawley rats were instrumented for chronic recording of sleep by electroencephalographic and electromyographic monitoring. Respirations were measured by single chamber plethysmograph, and blood pressure and heart period were transduced by a telemetric implant. Each rat was polygraphically recorded for 6 hours on four occasions in random order, with recordings for an individual animal separated by at least 3 days. Fifteen minutes prior to each recording (0945 hours) each animal received a 1 ml/kg intraperitoneal bolus injection of one of four injectates: saline (control) or 0.03 mg/kg, 0.3 mg/kg, or 3 mg/kg of GR79236. The study was a repeated-measures balanced design such that each animal was recorded exactly once for each injectate. The rate of spontaneous apneas (pauses > 2.5 seconds) was significantly reduced during all sleep stages by all doses of GR79236. At the highest dose, apnea index was reduced by over 70% in both non-rapid eye movement (NREM) and rapid eye movement (REM) sleep. In contrast, GR79236 had no effect on sleep stage volumes or blood pressure at any dose tested. Heart rate and core temperature were reduced only at the highest dose (3 mg/kg). We conclude that the adenosine A1 receptor agonist GR79236 significantly suppresses apnea expression in all sleep stages at doses not associated with significant changes in sleep architecture, blood pressure, heart rate, or core temperature.     

Adenosine A1 receptor-mediated activation of phospholipase C in cultured astrocytes depends on the level of receptor expression

Adenosine A1 receptors induce an inhibition of adenylyl cyclase via G-proteins of the Gi/o family. In addition, simultaneous stimulation of A1 receptors and of receptor-mediated activation of phospholipase C (PLC) results in a synergistic potentiation of PLC activity. Evidence has accumulated that Gbetagamma subunits mediate this potentiating effect. However, an A1 receptor-mediated increase in extracellular glutamate was suggested to be responsible for the potentiating effect in mouse astrocyte cultures. We have investigated the synergistic activation of PLC by adenosine A1 and alpha1 adrenergic receptors in primary cultures of astrocytes derived from different regions of the newborn rat brain. It is reported here that (1) adenosine A1 receptor mRNA as well as receptor protein is present in astrocytes from all brain regions, (2) A1 receptor-mediated inhibition of adenylyl cyclase is of similar extent in all astrocyte cultures, (3) the A1 receptor-mediated potentiation of PLC activity requires higher concentrations of agonist than adenylyl cyclase inhibition and is dependent on the expression level of A1 receptor, and (4) the potentiating effect on PLC activity is unrelated to extracellular glutamate. Taken together, our data support the notion that betagamma subunits are the relevant signal transducers for A1 receptor-mediated PLC activation in rat astrocytes. Because of the lower affinity of betagamma, as compared with alpha subunits, more betagamma subunits are required for PLC activation. Therefore, only in cultures with higher levels of adenosine A1 receptors is the release of betagamma subunits via Gi/o activation sufficient to stimulate PLC. It is concluded that variation of the expression level of adenosine A1 receptors may be an important regulatory mechanism to control PLC activation via this receptor.     

Adenosine A2 receptor mediation of pre- and postsynaptic excitatory effects of adenosine in rat hippocampus in vitro

The poly(beta-hydroxybutyrate) (PHB) biosynthetic genes of Ralstonia eutropha that are organized in a single operon (phaCAB) have been cloned in Escherichia coli, where the expression of the genes in the wild-type pha operon from plasmid pTZ18U-PHB leads to the formation of 50-80% PHB/celldry mass when the cells are grown in Luria-Bertani medium supplemented with 1% glucose (w/v). In combination with the phaCAB genes, expression of cloned lysis gene E of bacteriophage PhiX174 from plasmid pSH2 has been used to release PHB granules produced in E. coli. It was shown that small PHB granules in a semiliquid stage are squeezed out of the cells through the E-lysis tunnel structure which is characterized by a small opening in the envelope with borders of fused inner and outer membranes. All envelope components remain intact after E-lysis and can be removed from the mixture of released PHB granules by density gradient centrifugation. In addition, a modified E-lysis procedure is described which enables the release of PHB from cell pellets in pure water or low ionic strength buffer. PHB granules in aqueous solution can be aggregated by divalent cations. Addition of glassmilk speeds up the agglomeration of PHB granules and binding to glass beads can either be used for collection or further purification of PHB in aqueous solutions.     

Alpha 1-adrenoceptor subtype involved in the positive and negative inotropic responses to phenylephrine in rat papillary muscle

OBJECTIVE: Neoadjuvant androgen ablation (NAAA) causes significant cytoarchitectural changes in both benign and malignant prostatic epithelial cells that may contribute to underdetection of prostate cancer capsular involvement and positive surgical margins. METHODS: The aim of this study is to determine the ability of cytokeratin immunohistochemistry to enhance the determination of pathologic stage of prostate cancer following NAAA. RESULTS: Cytokeratin AE1/AE3 immunohistochemistry identified 6 (27.3%), 15 (68.2%), 5 (22.7%), and 5 (22.7%) cases of organ-confined disease, capsule penetration, positive surgical margin, and seminal vesicle involvement, respectively, as compared with 10 (45.5%), 10 (45.5%), 3 (13.6%), and 5 (22.7%) cases by hematoxylin-eosin (H&E) staining, respectively. Two cases without detectable tumor by H&E staining had demonstrable residual tumor by cytokeratin immunohistochemical staining. CONCLUSIONS: Cytokeratin immunohistochemistry revealed more extensive intracapsular, capsular, and extracapsular tumor involvement and higher rate of positive surgical margin than did conventional H&E staining. Therefore, the beneficial pathologic effects of NAAA observed may, in part, be attributable to the artifact of observation.     

The negative inotropic effect of beta3-adrenoceptor stimulation is mediated by activation of a nitric oxide synthase pathway in human ventricle

Beta1- and beta2-adrenoceptors in heart muscle cells mediate the catecholamine-induced increase in the force and frequency of cardiac contraction. Recently, in addition, we demonstrated the functional expression of beta3-adrenoceptors in the human heart. Their stimulation, in marked contrast with that of beta1- and beta2-adrenoceptors, induces a decrease in contractility through presently unknown mechanisms. In the present study, we examined the role of a nitric oxide (NO) synthase pathway in mediating the beta3-adrenoceptor effect on the contractility of human endomyocardial biopsies. The negative inotropic effects of a beta3-adrenoceptor agonist, BRL 37344, and also of norepinephrine in the presence of alpha- and beta1-2-blockade were inhibited both by a nonspecific blocker of NO, methylene blue, and two NO synthase (NOS) inhibitors, L-N-monomethyl-arginine and L-nitroarginine-methyl ester. The effect of the NOS inhibitors was reversed by an excess of L-arginine, the natural substrate of NOS, but not by D-arginine. Moreover, the effects of the beta3-adrenoceptor agonist on contractility were associated with parallel increases in the production of NO and intracellular cGMP, which were also inhibited by NOS inhibitors. Immunohistochemical staining of human ventricular biopsies showed the expression of the endothelial constitutive (eNOS), but not the inducible (iNOS) isoform of NOS in both ventricular myocytes and endothelial cells. These results demonstrate that beta3-adrenoceptor stimulation decreases cardiac contractility through activation of an NOS pathway. Changes in the expression of this pathway may alter the balance between positive and negative inotropic effects of catecholamines on the heart potentially leading to myocardial dysfunction.     

Adenosine A2A receptor antagonists as new agents for the treatment of Parkinson's disease

There is now good reason to believe that blockade of the adenosine A2A receptor could be of value in the treatment of Parkinson's disease. Peter J. Richardson, Hiroshi Kase and Peter G. Jenner review the actions of this receptor in the striatum, emphasizing its ability to modulate the neuronal activity of striatal GABA-releasing output neurones, and showing that recently developed A2A receptor antagonists are capable of reducing the disabling effects of nigral cell degeneration in primates. They conclude that such antagonists may be useful as novel therapeutic agents for the treatment of Parkinson's disease.     

Negative chronotropic and inotropic effects exerted by diadenosine hexaphosphate (AP6A) via A1-adenosine receptors

1. Diadenosine hexaphosphate (AP6A) exerts vasoconstrictive effects. The purpose of this study was to investigate whether AP6A has any effect on cardiac function. 2. The effects of AP6A (0.1-100 microM) on cardiac contractility and frequency were studied in guinea-pig and human isolated cardiac preparations. Furthermore, the effects of AP6A on the amplitude of the L-type calcium current, on the adenosine 3':5'-cyclic monophosphate (cyclic AMP) content and on the phosphorylation of regulatory phosphoproteins, i.e. phospholamban and troponin inhibitor, were investigated in guinea-pig isolated ventricular myocytes. 3. In isolated spontaneously beating right atria of the guinea-pig AP6A exerted a negative chronotropic effect and reduced the rate of contraction maximally by 35% (IC20 = 35 microM). 4. In isolated electrically driven left atria of the guinea-pig AP6A exerted a negative inotropic effect and reduced force of contraction maximally by 23% (IC20 = 70 microM). 5. In isolated electrically driven papillary muscles of the guinea-pig AP6A alone was ineffective, but attenuated isoprenaline-stimulated force of contraction maximally by 23% (IC20 = 60 microM). Furthermore, AP6A attenuated the relaxant effect of isoprenaline. 6. In human isolated electrically driven ventricular preparations AP6A alone was ineffective, but attenuated isoprenaline-stimulated force of contraction by maximally 42% (IC20 = 18 microM). Moreover, AP6A attenuated the relaxant effect of isoprenaline. 7. All these effects of AP6A were abolished by the selective A1-adenosine receptor antagonist 1,3-dipropyl-cyclopentyl-xanthine (DPCPX, 0.3 microM), whereas the M-cholinoceptor antagonist atropine (10 microM) and the P2-purinoceptor antagonist suramin (300 microM) failed to abolish the effects of AP6A. 8. AP6A 100 microM had no effect on the amplitude of the L-type calcium current, but attenuated isoprenaline-stimulated L-type calcium current. The maximum of the current-voltage relationship (I-V curve) was shifted to the left by isoprenaline and additional application of AP6A shifted the I-V curve back to the right to the control value. The phosphorylation state of phospholamban and the troponin inhibitor was unchanged by AP6A alone, but was markedly attenuated by AP6A in the presence of isoprenaline. Cyclic AMP levels remained unchanged by AP6A, even after stimulation with isoprenaline. 9. In summary, AP6A exerts negative chronotropic and inotropic effects in guinea-pig and human cardiac preparations. These effects are mediated via A1-adenosine receptors as all effects were sensitive to the selective A1-adenosine receptor antagonist DPCPX. Furthermore, the effects of AP6A on cyclic AMP levels, protein phosphorylation and the L-type calcium current are in accordance with stimulation of A1-adenosine receptors.     

Role of PKC in the effects of alpha1-adrenergic stimulation on Ca2+ transients, contraction and Ca2+ current in guinea-pig ventricular myocytes

The effects of alpha1-adrenoceptor stimulation on intracellular Ca2+ transients, contractility and L-type Ca2+ current (ICa,L) were studied in single cells isolated from ventricles of guinea-pig hearts. The aim of our study was to elucidate the mechanisms of the positive inotropic effect of alpha1-adrenergic stimulation by focussing on the role of protein kinase C (PKC). Phenylephrine, an alpha1-adrenergic agonist, at concentrations of 50-100 microM elicited a biphasic inotropic response: a transient negative inotropic response (22.9+/-6.0% of control) followed by a sustained positive inotropic response (61.0+/-8.4%, mean+/-SE, n=12). The Ca2+ transient decreased by 10.2+/-3.9% during the negative inotropic phase, while it increased by 67.7+/-10% (n=12) during the positive inotropic phase. These effects were inhibited by prazosin (1 microM), a alpha1-adrenergic antagonist. Phenylephrine increased the ICa,L by 60.8+/-21% (n=5) during the positive inotropic phase. To determine whether activation of PKC is responsible for the increases in Ca2+ transients, contractile amplitude and ICa,L during alpha1-adrenoceptor stimulation, we tested the effects of 4beta-phorbol 12-myristate 13-acetate (PMA), a PKC activator, and of bisindolylmaleimide I (GF109203X) and staurosporine, both of which are PKC inhibitors. PMA mimicked phenylephrine's effects on Ca2+ transients, contractile amplitude and ICa,L. PMA (100 nM) increased the Ca2+ transient, contractile amplitude and ICa,L by 131+/-17%, 137+/-25% (n=8), and 81.1+/-26% (n=5), respectively. Prior exposure to GF109203X (1 microM) or staurosporine (10 nM) prevented the phenylephrine-induced increases in Ca2+ transients, contractile amplitude and ICa,L. Our study suggests that during alpha1-adrenoceptor stimulation increase in ICa,L via PKC causes an increase in Ca2+ transients and thereby in the contractile force of the ventricular myocytes.     

Age-dependent decrease in the negative inotropic effect of carbachol on isolated human right atrium

On isolated, electrically driven human right atrial strips, carbachol (10(-8)-10(-3) M) concentration-dependently decreased force of contraction prestimulated with 1 microM forskolin; maximal negative inotropic effects of carbachol (10(-6)-3 x 10(-6) M), however, were in atria from patients aged < 25 years (mean age: 16.8 +/- 2.0 years, n = 9) significantly larger than in patients aged 50-69 years (mean age: 62.5 +/- 0.7 years, n = 33) and were further decreased in patients aged > 70 years (mean age: 73.8 +/- 0.6 years, n = 11). We conclude that, in human right atrium, the recently described age-dependent decrease in muscarinic M2 receptor density is accompanied by a decrease in negative inotropic effects.     

Application of mechanical restitution: variation of inotropic effects of vagal stimulation or verapamil administration during irregular cardiac rhythm

The effects of verapamil administration or vagal stimulation on the mechanical restitution curve (MRC) were studied in order to better understand the modulation of left ventricular (LV) function by interventions that lower the ventricular rate of atrial fibrillation. The MRC and the postextrasystolic MRC were obtained in 11 dogs using peak single beat elastance (Emax). The MRC was fitted by a monoexponential curve. Vagal stimulation or verapamil administration decreased the peak of the MRC and right-shifted the MRC. The postextrasystolic MRC was located upward compared with the control MRC, and was shifted downward by vagal stimulation or verapamil administration. If interventions having a negative inotropic effect effectively slow a rapid heart rate, the net effect of the ventricular contractile state may not always be negative. It was concluded that the MRC is useful in understanding LV contractility during irregular rhythm, especially when assessing the net effect of the negative dromotropic and inotropic action of antiarrhythmic drugs.     

Adenosine monophosphate as a mediator of ATP effects at P1 purinoceptors

1. When perfused with a medium containing no added magnesium and 4-aminopyridine (4AP) (50 microM) hippocampal slices generated epileptiform bursts of an interictal nature. We have shown in a previous study that adenosine 5'-triphosphate (ATP) depressed epileptiform activity and that this effect was blocked by the adenosine A1 receptor antagonist cyclopentyltheophylline but was not affected by adenosine deaminase. This implied that ATP might act indirectly at P1 receptors or at a xanthine-sensitive P2 receptor. The aim of the present study was to investigate further the action of ATP on epileptiform activity. 2. ATP can be metabolized by ecto-nucleotidases to adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP) and adenosine, respectively. Each of these metabolites can activate receptors in its own right: P2 receptors for ADP and P1 receptors for AMP and adenosine. 3. We now show that both AMP and ATP (50 microM) significantly decrease epileptiform discharge rate in a rapid and reversible manner. 5'Adenylic acid deaminase (AMP deaminase, AMPase) (0.2 u ml(-1)), when perfused alone did not significantly alter the discharge rate over the 10 min superfusion period used for drug application. When perfused concurrently with AMP (50 microM), AMP deaminase prevented the depressant effect of AMP on discharge rate. 4. AMP deaminase, at a concentration of 0.2 u ml(-1) which annulled the effect of AMP (50 microM), prevented the inhibitory activity of ATP (50 microM). A higher concentration of ATP (200 microM) depressed the frequency of spontaneous bursts to approximately 30% control and this response was also prevented by AMP deaminase. 5. Superfusion of the slices with 5'-nucleotidase also prevented the inhibitory activity of ATP on epileptiform discharges. 6. The results suggest that AMP mediates the inhibitory effects of ATP on epileptiform activity, a conclusion which can explain the earlier finding that cyclopentyltheophylline but not adenosine deaminase inhibited the effect of ATP. A corollary to this is that, when examining the pharmacology of ATP, care must be taken to inactivate AMP with AMP deaminase, as well as adenosine with adenosine deaminase, before a direct action of ATP on P1 receptors can be postulated. Failure to do so may have led to erroneous conclusions in some previous studies of nucleotide activity on nucleotide receptors.