Search

N-methyl-D-aspartate (NMDA) receptor antagonists are widely used to pharmacologically model schizophrenia and have been recently established in the treatment of treatment-resistant major depression demonstrating that the pharmacology of this substance class is complex. Cortical gamma oscillations, a rhythmic neuronal activity associated with cognitive processes, are increased in schizophrenia and deteriorated in depressive disorders and are increasingly used as biomarker in these neuropsychiatric diseases. The opposite use of NMDA receptor antagonists in schizophrenia and depression raises the question how their effects are in accordance with the observed disease pathophysiology and if these effects show a consequent sex-specificity. In this study in rats, we investigated the effects of subchronic (14 days) intraperitoneal injections of the NMDA receptor antagonist MK-801 at a subanesthetic daily dose of 0.2 mg/kg on the behavioral phenotype of adult female and male rats and on pharmacologically induced gamma oscillations measured ex vivo from the hippocampus. We found that MK-801 treatment leads to impaired recognition memory in the novel object recognition test, increased stereotypic behavior and reduced grooming, predominantly in female rats. MK-801 also increased the peak power of hippocampal gamma oscillations induced by kainate or acetylcholine only in female rats, without affecting the peak frequency of the oscillations. The findings indicate that blockade of NMDA receptors enhances gamma oscillations predominantly in female rats and this effect is associated with behavioral changes in females. The results are in accordance with clinical electrophysiological findings and highlight the importance of hippocampal gamma oscillations as a biomarker in schizophrenia and depression. [ABSTRACT FROM AUTHOR]

Background and Purpose: P2X4 receptors (P2X4R) are ligand gated cation channels that are activated by extracellular ATP released by neurons and glia. The receptors are widely expressed in the brain and have fractional calcium currents comparable with NMDA receptors. Although P2X4Rs have been reported to modulate synaptic transmission and plasticity, their involvement in shaping neuronal network activity remains to be elucidated. Experimental Approach: We investigated the effects of P2X receptors at network and synaptic level using local field potential electrophysiology, whole cell patch clamp recordings and calcium imaging in fast spiking parvalbumin positive interneurons (PVINs) in rat and mouse hippocampal slices. The stable ATP analogue ATPγS, selective antagonists and P2X4R knockout mice were used. Key Results: The P2XR agonist ATPγS reversibly decreased the power of gamma oscillations. This inhibition could be antagonized by the selective P2X4R antagonist PSB‐12062 and was not observed in P2X4−/− mice. The phasic excitatory inputs of CA3 PVINs were one of the main regulators of the gamma power. Associational fibre compound excitatory postsynaptic currents (cEPSCs) in CA3 PVINs were inhibited by P2X4R activation. This effect was reversible, dependent on intracellular calcium and dynamin‐dependent internalization of AMPA receptors. Conclusions and Implications: The results indicate that P2X4Rs are an important source of dendritic calcium in CA3 PVINs, thereby regulating excitatory synaptic inputs onto the cells and presumably the state of gamma oscillations in the hippocampus. P2X4Rs represent an effective target to modulate hippocampal network activity in pathophysiological conditions such as Alzheimer's disease and schizophrenia. [ABSTRACT FROM AUTHOR]

Myelination enhances the conduction velocity of action potentials (AP) and increases energy efficiency. Thick myelin sheaths are typically found on large-distance axonal connections or in fast-spiking interneurons, which are critical for synchronizing neuronal networks during gamma-band oscillations. Loss of myelin sheath is associated with multiple alterations in axonal architecture leading to impaired AP propagation. While numerous studies are devoted to the effects of demyelination on conduction velocity, the metabolic effects and the consequences for network synchronization have not been investigated. Here we present a unifying computational model for electrophysiology and metabolism of the myelinated axon. The computational model suggested that demyelination not only decreases the AP speed but AP propagation in demyelinated axons requires compensatory processes like mitochondrial mass increase and a switch from saltatory to continuous propagation to rescue axon functionality at the cost of reduced AP propagation speed and increased energy expenditure. Indeed, these predictions were proven to be true in a culture model of demyelination where the pharmacologically-induced loss of myelin was associated with increased oxygen consumption rates, and a significant broadening of bandwidth as well as a decrease in the power of gamma oscillations. [ABSTRACT FROM AUTHOR]

Ion channels activated around the subthreshold membrane potential determine the likelihood of neuronal firing in response to synaptic inputs, a process described as intrinsic neuronal excitability. Long-term plasticity of chemical synaptic transmission is traditionally considered the main cellular mechanism of information storage in the brain; however, voltage- and calcium-activated channels modulating the inputs or outputs of neurons are also subjects of plastic changes and play a major role in learning and memory formation. Gamma oscillations are associated with numerous higher cognitive functions such as learning and memory, but our knowledge of their dependence on intrinsic plasticity is by far limited. Here we investigated the roles of potassium and calcium channels activated at near subthreshold membrane potentials in cholinergically induced persistent gamma oscillations measured in the CA3 area of rat hippocampal slices. Among potassium channels, which are responsible for the afterhyperpolarization in CA3 pyramidal cells, we found that blockers of SK (KCa2) and KV7.2/7.3 (KCNQ2/3), but not the BK (KCa1.1) and IK (KCa3.1) channels, increased the power of gamma oscillations. On the contrary, activators of these channels had an attenuating effect without affecting the frequency. Pharmacological blockade of the low voltage-activated T-type calcium channels (CaV3.1–3.3) reduced gamma power and increased the oscillation peak frequency. Enhancement of these channels also inhibited the peak power without altering the frequency of the oscillations. The presented data suggest that voltage- and calcium-activated ion channels involved in intrinsic excitability strongly regulate the power of hippocampal gamma oscillations. Targeting these channels could represent a valuable pharmacological strategy against cognitive impairment. [ABSTRACT FROM AUTHOR]

Extracellular purines are important signaling molecules involved in numerous physiological and pathological processes via the activation of P2 receptors. Information about the spatial and temporal P2 receptor (P2R) expression and its regulation remains crucial for the understanding of the role of P2Rs in health and disease. To identify cells carrying P2X2Rs in situ, we have generated BAC transgenic mice that express the P2X2R subunits as fluorescent fusion protein (P2X2-TagRFP). In addition, we generated a BAC P2Y1R TagRFP reporter mouse expressing a TagRFP reporter for the P2RY1 gene expression. We demonstrate expression of the P2X2R in a subset of DRG neurons, the brain stem, the hippocampus, as well as on Purkinje neurons of the cerebellum. However, the weak fluorescence intensity in our P2X2R-TagRFP mouse precluded tracking of living cells. Our P2Y1R reporter mice confirmed the widespread expression of the P2RY1 gene in the CNS and indicate for the first time P2RY1 gene expression in mouse Purkinje cells, which so far has only been described in rats and humans. Our P2R transgenic models have advanced the understanding of purinergic transmission, but BAC transgenic models appeared not always to be straightforward and permanent reliable. We noticed a loss of fluorescence intensity, which depended on the number of progeny generations. These problems are discussed and may help to provide more successful animal models, even if in future more versatile and adaptable nuclease-mediated genome-editing techniques will be the methods of choice. [ABSTRACT FROM AUTHOR]

The actin binding protein drebrin plays a key role in dendritic spine formation and synaptic plasticity. Decreased drebrin protein levels have been observed in temporal lobe epilepsy, suggesting the involvement of drebrin in the disease. Here we investigated the effect of drebrin knockout on physiological and pathophysiological neuronal network activities in mice by inducing gamma oscillations, involved in higher cognitive functions, and by analyzing pathophysiological epileptiform activity. We found that loss of drebrin increased the emergence of spontaneous gamma oscillations suggesting an increase in neuronal excitability when drebrin is absent. Further analysis showed that although the kainate-induced hippocampal gamma oscillations were unchanged in drebrin deficient mice, seizure like events measured in the entorhinal cortex appeared earlier and more frequently. The results suggest that while drebrin is not essential for normal physiological network activity, it helps to protect against the formation of seizure like activities during pathological conditions. The data indicate that targeting drebrin function could potentially be a preventive or therapeutic strategy for epilepsy treatment. [ABSTRACT FROM AUTHOR]

Background and Purpose: Gamma oscillations are fast rhythmic fluctuations of neuronal network activity ranging from 30 to 90 Hz that establish a precise temporal background for cognitive processes such as perception, sensory processing, learning, and memory. Alterations of gamma oscillations have been observed in schizophrenia and are suggested to play crucial roles in the generation of positive, negative, and cognitive symptoms of the disease.Experimental Approach: In this study, we investigated the effects of the novel antipsychotic cariprazine, a D3 -preferring dopamine D3 /D2 receptor partial agonist, on cholinergically induced gamma oscillations in rat hippocampal slices from treatment-naïve and MK-801-treated rats, a model of acute first-episode schizophrenia.Key Results: The D3 receptor-preferring agonist pramipexole effectively decreased the power of gamma oscillations, while the D3 receptor antagonist SB-277011 had no effect. In treatment-naïve animals, cariprazine did not modulate strong gamma oscillations but slightly improved the periodicity of non-saturated gamma activity. Cariprazine showed a clear partial agonistic profile at D3 receptors at the network level by potentiating the inhibitory effects when the D3 receptor tone was low and antagonizing the effects when the tone was high. In hippocampal slices of MK-801-treated rats, cariprazine allowed stabilization of the aberrant increase in gamma oscillation power and potentiated resynchronization of the oscillations.Conclusion and Implications: Data from this study indicate that cariprazine stabilizes pathological hippocampal gamma oscillations, presumably by its partial agonistic profile. The results demonstrate in vitro gamma oscillations as predictive biomarkers to study the effects of antipsychotics preclinically at the network level. [ABSTRACT FROM AUTHOR]

Epilepsy is characterized by the regular occurrence of seizures, which follow a stereotypical sequence of alterations in the electroencephalogram. Seizures are typically a self limiting phenomenon, concluding finally in the cessation of hypersynchronous activity and followed by a state of decreased neuronal excitability which might underlie the cognitive and psychological symptoms the patients experience in the wake of seizures. Many efforts have been devoted to understand how seizures spontaneously stop in hope to exploit this knowledge in anticonvulsant or neuroprotective therapies. Besides the alterations in ion-channels, transmitters and neuromodulators, the successive build up of disturbances in energy metabolism have been suggested as a mechanism for seizure termination. Energy metabolism and substrate supply of the brain are tightly regulated by different mechanisms called neurometabolic and neurovascular coupling. Here we summarize the current knowledge whether these mechanisms are sufficient to cover the energy demand of hypersynchronous activity and whether a mismatch between energy need and supply could contribute to seizure control. [ABSTRACT FROM AUTHOR]

Objective The need for alternative pharmacologic strategies in treatment of epilepsies is pressing for about 30% of patients with epilepsy who do not experience satisfactory seizure control with present treatments. In temporal lobe epilepsy ( TLE) even up to 80% of patients are pharmacoresistant, and surgical resection of the ictogenic tissue is only possible for a minority of TLE patients. In this study we investigate purinergic modulation of drug-resistant seizure-like events ( SLEs) in human temporal cortex slices. Methods Layer V/ VI field potentials from a total of 77 neocortical slices from 17 pharmacoresistant patients were recorded to monitor SLEs induced by application of 8 mM [K+] and 50 μ m bicuculline. Results Activating A1 receptors with a specific agonist completely suppressed SLEs in 73% of human temporal cortex slices. In the remaining slices, incidence of SLEs was markedly reduced. Because a subportion of slices can be pharmacosensitive, we tested effects of an A1 agonist, in slices insensitive to a high dose of carbamazepine (50 μ m). Also in these cases the A1 agonist was equally efficient. Moreover, ATP and adenosine blocked or modulated SLEs, an effect mediated not by P2 receptors but rather by adenosine A1 receptors. Significance Selective activation of A1 receptors mediates a strong anticonvulsant action in human neocortical slices from pharmacoresistant patients. We propose that our human slice model of seizure-like activity is a feasible option for future studies investigating new antiepileptic drug ( AED) candidates. [ABSTRACT FROM AUTHOR]

ATP released from neurons and astrocytes during neuronal activity or under pathophysiological circumstances is able to influence information flow in neuronal circuits by activation of ionotropic P2X andmetabotropic P2Y receptors and subsequentmodulation of cellular excitability, synaptic strength, and plasticity. In the present paper we review cellular and network effects of P2Y receptors in the brain.We show that P2Y receptors inhibit the release of neurotransmitters,modulate voltage- and ligand-gated ion channels, and differentially influence the induction of synaptic plasticity in the prefrontal cortex, hippocampus, and cerebellum. The findings discussed heremay explain howP2Y1 receptor activation during brain injury, hypoxia, inflammation, schizophrenia, orAlzheimer’s disease leads to an impairment of cognitive processes. Hence, it is suggested that the blockade of P2Y1 receptors may have therapeutic potential against cognitive disturbances in these states. [ABSTRACT FROM AUTHOR]

Cortical gamma oscillations are associated with cognitive processes and are altered in several neuropsychiatric conditions such as schizophrenia and Alzheimer's disease. Since dopamine D3 receptors are possible targets in treatment of these conditions, it is of great importance to understand their role in modulation of gamma oscillations. The effect of D3 receptors on gamma oscillations and the underlying cellular mechanisms were investigated by extracellular local field potential and simultaneous intracellular sharp micro-electrode recordings in the CA3 region of the hippocampus in vitro. D3 receptors decreased the power and broadened the bandwidth of gamma oscillations induced by acetylcholine or kainate. Blockade of the D3 receptors resulted in faster synchronization of the oscillations, suggesting that endogenous dopamine in the hippocampus slows down the dynamics of gamma oscillations by activation of D3 receptors. Investigating the underlying cellular mechanisms for these effects showed that D3 receptor activation decreased the rate of action potentials (APs) during gamma oscillations and reduced the precision of the AP phase coupling to the gamma cycle in CA3 pyramidal cells. The results may offer an explanation how selective activation of D3 receptors may impair cognition and how, in converse, D3 antagonists may exert pro-cognitive and antipsychotic effects. [ABSTRACT FROM AUTHOR]

Stressful experiences do not only cause peripheral changes in stress hormone levels, but also affect central structures such as the hippocampus, implicated in spatial orientation, stress evaluation, and learning and memory. It has been suggested that formation of memory traces is dependent on hippocampal gamma oscillations observed during alert behaviour and rapid eye movement sleep. Furthermore, during quiescent behaviour, sharp wave-ripple ( SW- R) activity emerges. These events provide a temporal window during which reactivation of memory ensembles occur. We hypothesized that stress-responsive modulators, such as corticosterone ( CORT), corticotropin-releasing factor ( CRF) and the neurosteroid 3α, 21-dihydroxy-5α-pregnan-20-one ( THDOC) are able to modulate gamma oscillations and SW- Rs. Using in vitro hippocampal slices, we studied acute and subacute (2 h) impact of these agents on gamma oscillations in area cornu ammonis 3 of the ventral hippocampus induced by acetylcholine (10 μ m) combined with physostigmine (2 μ m). CORT increased the gamma oscillations in a dose-dependent fashion. This effect was mediated by glucocorticoid receptors. Likewise, CRF augmented gamma oscillations via CRF type 1 receptor. Lastly, THDOC was found to diminish cholinergic gamma oscillations in a dose-dependent manner. Neither CORT, CRF nor THDOC modulated gamma power when pre-applied for 1 h, 2 h before the induction of gamma oscillations. Interestingly, stress-related neuromodulators had rather mild effects on spontaneous SW- R compared with their effects on gamma oscillations. These data suggest that the alteration of hippocampal gamma oscillation strength in vitro by stress-related agents is an acute process, permitting fast adaptation to new attention-requiring situations in vivo. [ABSTRACT FROM AUTHOR]

Background and Purpose: Disturbed cortical gamma band oscillations (30-80 Hz) have been observed in schizophrenia: positive symptoms of the disease correlate with an increase in gamma oscillation power, whereas negative symptoms are associated with a decrease.Experimental Approach: Here we investigated the effects of first and second generation antipsychotics (FGAs and SGAs, respectively) on gamma oscillations. The FGAs haloperidol, flupenthixol, chlorpromazine, chlorprothixene and the SGAs clozapine, risperidone, ziprasidone, amisulpride were applied on gamma oscillations induced by acetylcholine and physostigmine in the CA3 region of rat hippocampal slices.Key Results: Antipsychotics inhibited the power of gamma oscillations and increased the bandwidth of the gamma band. Haloperidol and clozapine had the highest inhibitory effects. To determine which receptor is responsible for the alterations in gamma oscillations, the effects of the antipsychotics were plotted against their pK(i) values for 19 receptors and analysed for correlation. Our results indicated that 5-HT(3) receptors have an enhancing effect on gamma oscillations whereas dopamine D(3) receptors inhibit them. To test this prediction, m-chlorophenylbiguanide, PD 128907 and CP 809101, selective agonists at 5-HT(3) , D(3) and 5-HT(2C) receptors were applied and revealed that 5-HT(3) receptors indeed enhanced the gamma power whereas D(3) receptors reduced it. As predicted, 5-HT(2C) receptors had no effects on gamma oscillations.Conclusion and Implications: Our data suggest that antipsychotics alter hippocampal gamma oscillations by interacting with 5-HT(3) and dopamine D(3) receptors. Moreover, a correlation of receptor affinities with the biological effects can be used to predict targets for the pharmacological effects of multi-target drugs. [ABSTRACT FROM AUTHOR]

BACKGROUND AND PURPOSE Disturbed cortical gamma band oscillations (30-80 Hz) have been observed in schizophrenia: positive symptoms of the disease correlate with an increase in gamma oscillation power, whereas negative symptoms are associated with a decrease. EXPERIMENTAL APPROACH Here we investigated the effects of first and second generation antipsychotics (FGAs and SGAs, respectively) on gamma oscillations. The FGAs haloperidol, flupenthixol, chlorpromazine, chlorprothixene and the SGAs clozapine, risperidone, ziprasidone, amisulpride were applied on gamma oscillations induced by acetylcholine and physostigmine in the CA3 region of rat hippocampal slices. KEY RESULTS Antipsychotics inhibited the power of gamma oscillations and increased the bandwidth of the gamma band. Haloperidol and clozapine had the highest inhibitory effects. To determine which receptor is responsible for the alterations in gamma oscillations, the effects of the antipsychotics were plotted against their p Ki values for 19 receptors and analysed for correlation. Our results indicated that 5-HT3 receptors have an enhancing effect on gamma oscillations whereas dopamine D3 receptors inhibit them. To test this prediction, m-chlorophenylbiguanide, PD 128907 and CP 809101, selective agonists at 5-HT3, D3 and 5-HT2C receptors were applied and revealed that 5-HT3 receptors indeed enhanced the gamma power whereas D3 receptors reduced it. As predicted, 5-HT2C receptors had no effects on gamma oscillations. CONCLUSION AND IMPLICATIONS Our data suggest that antipsychotics alter hippocampal gamma oscillations by interacting with 5-HT3 and dopamine D3 receptors. Moreover, a correlation of receptor affinities with the biological effects can be used to predict targets for the pharmacological effects of multi-target drugs. [ABSTRACT FROM AUTHOR]

Purpose: Adenosine is considered an endogenous anticonvulsant. However, much less is known about the putative effects of its precursor, ATP, on epilepsy. Therefore, we tested whether ATP and its receptors are able to modulate epileptiform activity in the medial entorhinal cortex of the rat. Methods: Recurrent epileptiform discharges (REDs) were induced by elevating extracellular potassium concentration combined with application of bicuculline in brain slices from naive and pilocarpine-treated chronic epileptic rats. Field potentials were recorded from layer V/VI of the medial entorhinal cortex. Key Findings: REDs in slices from naive animals had a higher incidence and a shorter duration than in slices from chronic epileptic animals. Exogenous application of ATP reversibly reduced the incidence of REDs in naive and chronic epileptic slices via activation of adenosine A1 receptors without discernible P2 receptor effects. This effect was stronger in slices from chronic epileptic rats. In slices from naive rats, the P2X7 receptor antagonist A 740003 slightly but significantly reduced the amplitude of slow field potentials of REDs. In slices from chronic epileptic rats, none of the P2 receptor antagonists affected the parameters of REDs. Significance: Our results suggest that endogenously released ATP differentially modulates REDs by activation of A1 and P2X7 receptors. Although it has a minor proepileptic effect by direct activation of P2X7 receptors, its metabolite adenosine reduces the epileptiform activity via activation of A1 receptors. The exact effect of ATP on neural activity depends on the actual activity of ectonucleotidases and the expression level of the purinergic receptors, which both alter during epileptogenesis. In addition, our data suggest that P2X7 receptor antagonists have a minor antiepileptic effect. [ABSTRACT FROM AUTHOR]

Ionotropic P2X and metabotropic P2Y receptors interact with a number of macromolecules in the cell membrane which may contribute to their functional plasticity. P2X receptors are homomeric or heteromeric assemblies of three subunits. P2Y receptors may form oligomeric complexes either with the same or with other P2Y receptor types. Although the signalling mechanism of P2X receptor channels is fast (within milliseconds) and relatively simple, by originating from the opening of an ion channel permeable to mono- and divalent cations, various macromolecules may modify the trafficking of these receptors to and from the cell membrane, as well as their activation and desensitization kinetics, and the possible opening of membrane pores induced by long-lasting exposure to agonists. P2X and Cys-loop receptors may physically interact with each other, resulting in mutual current occlusion. Heteromeric P2Y receptors may, via Gs, Gq/11 or Gi/o protein-coupling and activation of the respective transduction mechanisms, mediate responses in the range of a few seconds. However, P2Y receptors may also interact with the signalling cascade of, e.g. receptor tyrosine kinases, and thereby mediate responses on a much slower time scale (within hours to days). In addition, P2Y receptors may interact with small, homomeric G proteins, integrins, and PDZ proteins. Eventually, P2Y receptors may cross-talk via Gα-dependent signalling with other G protein-coupled receptors and via Gβγ (or indirectly Gα)-dependent signalling with various ion channels. Thus, the activation of P2X and P2Y receptors by extracellular adenosine triphosphate/adenosine diphosphate or uridine triphosphate/uridine diphosphate may trigger specific chains of events which interact at the level of the individual elements both with each other and with the transduction mechanisms of other receptors, creating a huge diversity of the possible effects. [ABSTRACT FROM AUTHOR]

Mixed striatal cell cultures containing neurons and glial cells were grown either in neurobasal medium (NBM) or Dulbecco's modified Eagle's medium (DMEM). Whole-cell patch-clamp recordings indicated that, if at all, only a single, low amplitude spike was evoked shortly after starting the injection of a depolarizing current pulse into NBM neurons. In contrast, DMEM neurons fired series of high amplitude action potentials, without apparent spike frequency adaptation. The possible reason for the observed action potential failure in NBM neurons was a low density of Na+ channels per unit of membrane surface area. However, both in NBM and DMEM neurons, ATP did not induce inward current responses via P2X receptor-channels, although GABAA and N-methyl-D-aspartate (NMDA) receptor-channels could be activated by muscimol and NMDA, respectively. Ca2+ imaging experiments by means of the Fura-2 method were utilized to measure intracellular Ca2+ ([Ca2+]i) in neurons and glial cells. NBM, but not DMEM neurons responded to ATP with [Ca2+]i transients; glial cells grown in either culture medium were equally sensitive to ATP. ATP caused an increase of [Ca2+]i by a mechanism only partly dependent on external Ca2+; the residual ATP effect was blocked by cyclopiazonic acid (CPA) and was therefore due to the release of Ca2+ from its intracellular pools. The receptor involved was characterized by P2 receptor antagonists (PPADS, MRS 2179, AR-C69931MX) and was found to belong to the P2Y1 subtype. CPA caused an early [Ca2+]i response due to release from intracellular storage sites, followed by a late [Ca2+]i response due to the influx of this cation from the extracellular space, probably triggered by the opening of store-operated channels (SOCs) in the plasma membrane. It is concluded that in partial analogy with the effect of CPA, ATP releases [Ca2+]i via the Gq/phospholipase C/inositoltrisphosphate (IP3) pathway, thereby opening SOCs. It is hypothesized that this effect of ATP may have an important role for the proliferation and migration of striatal neuronal progenitors. J. Cell. Physiol. 209: 81–93, 2006. © 2006 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

It is widely accepted that the most important ATP receptors involved in pain transmission belong to the P2X3 and P2X2/3 subtypes, selectively expressed in small diameter dorsal root ganglion (DRG) neurons. However, several types of the metabotropic ATP (P2Y) receptors have also been found in primary afferent neurons; P2Y1 and P2Y2 receptors are typically expressed in small, nociceptive cells. Here we review the results available on the involvement of P2Y receptors in the modulation of pain transmission. [ABSTRACT FROM AUTHOR]

Patch-clamp recordings from small-diameter rat dorsal root ganglion (DRG) neurons maintained in culture demonstrated preferential inhibition by ATP of high-voltage-activated, but not low-voltage-activated, Ca&sup2+; currents (I[subCa]). The rank order of agonist potency was UTP > ADP > ATP. ATP depressed the ω-conotoxin GVIA-sensitive N-type current only. Pyridoxal-5-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) and 2'-deoxy-N[sup6]-methyladenosine 3',5'-bisphosphate tetraammonium, two P2Y[sub1] receptor antagonists, almost abolished the ATP-induced inhibition. Both patch-clamp recordings and immunocytochemistry coupled with confocal laser microscopy indicated a colocalization of functional P2X[sub3] and P2Y[sub1] receptors on the same DRG neurons. Because the effect of ATP was inhibited by intracellular guanosine 5'-0-(2-thiodiphosphate) or by applying a strongly depolarizing prepulse, P2Y[sub1] receptors appear to block I[subCa] by a pathway involving the βγ subunit of a G[subq/11] protein. Less efficient buffering of the intracellular Ca&sup2+; concentration ([Ca&sup2+;][subi]) by reducing the intrapipette EGTA failed to interfere with the ATP effect. Fura-2 microfluorimetry suggested that ATP raised [Ca&sup2+;][subi] by a Gα-mediated release from intracellular pools and simultaneously depressed the high external potassium concentration-induced increase of [Ca&sup2+;][subi] by inhibiting I[subCa] via Gβγ. Adenosine 5'-0-(2-thiodiphosphate) inhibited dorsal root-evoked polysynaptic population EPSPs in the hemisected rat spinal cord and prolonged the nociceptive threshold on intrathecal application in the tail-flick assay. These effects were not antagonized by PPADS. Hence, P2Y receptor activation by ADP, which is generated by enzymatic degradation of ATP, may decrease the release of glutamate from DRG terminals in the spinal cord and thereby partly counterbalance the algogenic effected on ATP. [ABSTRACT FROM AUTHOR]

1: N-type Ca2+ channel modulation by an endogenous P2Y receptor was investigated by the whole-cell patch-clamp method in HEK 293 cells transfected with the functional rabbit N-type calcium channel. 2: The current responses (ICa(N)) to depolarizing voltage steps were depressed by ATP in a concentration-dependent manner. Inclusion of either guanosine 5'-O-(3-thiodiphosphate) or pertussis toxin into the pipette solution as well as a strongly depolarizing prepulse abolished the inhibitory action of ATP. 3: In order to identify the P2Y receptor subtype responsible for this effect, several preferential agonists and antagonists were studied. Whereas the concentration-response curves of ADP and adenosine 5'-O-(2-thiodiphosphate) indicated a higher potency of these agonists than that of ATP, a,ß-methylene ATP, UTP and UDP were considerably less active. The effect of ATP was abolished by the P2Y receptor antagonists suramin and N6-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-ß,?-dichloromethylene-ATP, but not by pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid, 2'deoxy-N6-methyladenosine-3',5'-diphosphate or 2-methylthio AMP. 4: Using reverse transcription and polymerase chain reaction, mRNA for the P2Y1, P2Y4, P2Y6, P2Y11 and P2Y13 receptor subtypes, but not the P2Y2, and P2Y12 subtypes, was detected in HEK 293 cells. 5: Immunocytochemistry confirmed the presence of P2Y1, and to a minor extent that of P2Y4, but not of P2Y2 receptors. 6: Hence, it is tempting to speculate that P2Y13 receptors may inhibit N-type Ca2+ channels via the ß? subunits of the activated Gi protein.British Journal of Pharmacology (2004) 141, 141-151. doi:10.1038/sj.bjp.0705588 [ABSTRACT FROM AUTHOR]

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper. [ABSTRACT FROM AUTHOR]

It has been proposed that P2X3 receptors possess a unique mechanism of agonist-induced conformational transitions. Recovery from ATP-induced desensitization was found to be very slow; during this period a special agonist binding site was supposed to be formed which should bind the agonist with high affinity and promote desensitization without activation. The authors supposed that this high affinity binding site is absent from non-activated receptors. The theory was supported by an unexpected outcome of an experiment in which a low concentration of agonist was applied at different phases during recovery from desensitization. The inhibition by a low concentration of agonist was stronger when it was applied during the early phase of recovery when more desensitized receptors were present. The authors used different agonists for initial desensitization and for prolonged perfusion at low concentration. We repeated the experiment on HEK 293 cells expressing human P2X3 receptors with the same results. However, when we used the same agonist at both concentrations the inhibition was stronger when the low concentration was applied during the late phase. Simulations revealed that formation of high affinity binding sites does not require any unique mechanism and can be readily described by an allosteric mechanism. Furthermore, they predict that the unexpected phenomenon can only occur when a rapidly dissociating drug is replaced by a slowly dissociating drug on the receptor. [ABSTRACT FROM AUTHOR]