Tanshinone II A sulfonate, but not tanshinone II A, acts as potent negative allosteric modulator of the human purinergic receptor P2X7.

Tanshinone II A sulfonate (TIIAS) was identified as a potent, selective blocker of purinergic receptor P2X7 in a compound library screen. In this study, a detailed characterization of the pharmacologic effects of TIIAS on P2X7 is provided. Because TIIAS is a derivative of tanshinone II A (TIIA) and both compounds have been used interchangeably, TIIA was included in some assays. Fluorometric and electrophysiologic assays were used to characterize effects of TIIAS and TIIA on recombinantly expressed human, rat, and mouse P2X7. Results were confirmed in human monocyte-derived macrophages expressing native P2X7. In all experiments, involvement of P2X7 was verified using established P2X7 antagonists. TIIAS, but not TIIA, reduces Ca(2+) influx via human P2X7 (hP2X7) with an IC50 of 4.3 µM. TIIAS was less potent at mouse P2X7 and poorly inhibited rat P2X7. Monitoring of YO-PRO-1 uptake confirmed these findings, indicating that formation of the hP2X7 pore is also suppressed by TIIAS. Electrophysiologic experiments revealed a noncompetitive mode of action. TIIAS time-dependently inhibits hP2X7 gating, possibly by binding to the intracellular domain of the receptor. Inhibition of native P2X7 in macrophages by TIIAS was confirmed by monitoring Ca(2+) influx, YO-PRO-1 uptake, and release of the proinflammatory cytokine interleukin-1ß. Fluorometric experiments involving recombinantly expressed rat P2X2 and human P2X4 were conducted and verified the compound's selectivity. Our data suggest that hP2X7 is a molecular target of TIIAS, but not of TIIA, a compound with different pharmacologic properties.

Positive allosteric modulation by ivermectin of human but not murine P2X7 receptors.

BACKGROUND AND PURPOSE: In mammalian cells, the anti-parasitic drug ivermectin is known as a positive allosteric modulator of the ATP-activated ion channel P2X4 and is used to discriminate between P2X4- and P2X7-mediated cellular responses. In this paper we provide evidence that the reported isoform selectivity of ivermectin is a species-specific phenomenon. EXPERIMENTAL APPROACH: Complementary electrophysiological and fluorometric methods were applied to evaluate the effect of ivermectin on recombinantly expressed and on native P2X7 receptors. A biophysical characterization of ionic currents and of the pore dilation properties is provided. KEY RESULTS: Unexpectedly, ivermectin potentiated currents in human monocyte-derived macrophages that endogenously express hP2X7 receptors. Likewise, currents and [Ca(2+) ](i) influx through recombinant human (hP2X7) receptors were potently enhanced by ivermectin at submaximal or saturating ATP concentrations. Since intracellular ivermectin did not mimic or prevent its activity when applied to the bath solution, the binding site of ivermectin on hP2X7 receptors appears to be accessible from the extracellular side. In contrast to currents through P2X4 receptors, ivermectin did not cause a delay in hP2X7 current decay upon ATP removal. Interestingly, NMDG(+) permeability and Yo-Pro-1 uptake were not affected by ivermectin. On rat or mouse P2X7 receptors, ivermectin was only poorly effective, suggesting a species-specific mode of action. CONCLUSIONS AND IMPLICATIONS: The data indicate a previously unrecognized species-specific modulation of human P2X7 receptors by ivermectin that should be considered when using this cell-biological tool in human cells and tissues.

Electrophysiological classification of P2X7 receptors in rat cultured neocortical astroglia.

BACKGROUND AND PURPOSE: P2X7 receptors are ATP-gated cation channels mediating important functions in microglial cells, such as the release of cytokines and phagocytosis. Electrophysiological evidence that these receptors also occur in CNS astroglia is rare and rather incomplete. EXPERIMENTAL APPROACH: We used whole-cell patch-clamp recordings to search for P2X7 receptors in astroglial-neuronal co-cultures prepared from the cerebral cortex of rats. KEY RESULTS: All the astroglial cells investigated responded to ATP with membrane currents, reversing around 0 mV. These currents could be also detected in isolated outside-out patch vesicles. The results of the experiments with the P2X [alpha,beta-methylene ATP and 2'-3'-O-(4-benzoyl) ATP] and P2Y receptor agonists [adenosine 5'-O-(2-thiodiphosphate), uridine 5'-diphosphate, uridine 5'-triphosphate (UTP) and UDP-glucose] suggested the involvement of P2X receptors in this response. The potentiation of ATP responses in a low divalent cation or alkaline bath, but not by ivermectin, made it likely that a P2X7 receptor is operational. Blockade of the ATP effect by the P2X7 antagonists Brilliant Blue G, calmidazolium and oxidized ATP corroborated this assumption. CONCLUSIONS AND IMPLICATIONS: Rat cultured cortical astroglia possesses functional P2X7 receptors. It is suggested that astrocytic P2X7 receptors respond to high local ATP concentrations during neuronal injury.

Increase of intracellular Ca2+ by P2X and P2Y receptor-subtypes in cultured cortical astroglia of the rat.

Astrocytes express purinergic receptors that are involved in glial-neuronal cell communication. Experiments were conducted to characterize the expression of functional P2X/P2Y nucleotide receptors in glial cells of mixed cortical cell cultures of the rat. The vast majority of these cells was immunopositive for glial fibrillary acidic protein (GFAP) and was considered therefore astrocyte-like; for the sake of simplicity they were termed "astroglia" throughout. Astroglia expressed predominantly P2X(4,6,7) as well as P2Y(1,2) receptor-subtypes. Less intensive immunostaining was also found for P2X(5) and P2Y(4,6,13,14) receptors. Pressure application of ATP and a range of agonists selective for certain P2X or P2Y receptor-subtypes caused a concentration-dependent increase of intracellular Ca(2+) ([Ca(2+)](i)). Of the agonists tested, only the P2X(1,3) receptor-selective alpha,beta-methylene ATP was ineffective. Experiments with Ca(2+)-free solution and cyclopiazonic acid, an inhibitor of the endoplasmic Ca(2+)-ATPase, indicated that the [Ca(2+)](i) response to most nucleotides, except for ATP and 2',3'-O-(benzoyl-4-benzoyl)-ATP, was due primarily to the release of Ca(2+) from intracellular stores. A Gprotein-mediated release of Ca(2+) is the typical signaling mechanism of various P2Y receptor-subtypes, whose presence was confirmed also by cross-desensitization experiments and by using selective antagonists. Thus, our results provide direct evidence that astroglia in mixed cortical cell cultures express functional P2Y (P2Y(1,2,6,14) and probably also P2Y(4)) receptors. Several unidentified P2X receptors, including P2X(7), may also be present, although they appear to only moderately participate in the regulation of [Ca(2+)](i). The rise of [Ca(2+)](i) is due in this case to the transmembrane flux of Ca(2+) via the P2X receptor-channel. In conclusion, P2Y rather than P2X receptor-subtypes are involved in modulating [Ca(2+)](i) of cultured astroglia and thereby may play an important role in cell-to-cell signaling.

Diabatic states from nodal structure conservation.

For a Hamiltonian H(q), given in a suitable set of basis states, we construct diabatic states from requiring conservation of their nodal structure. The diabatic states and energies are single-valued functions for an arbitrary number of parameters q equivalent to {q1,q2,...q(f)}. The method is illustrated for nucleons moving in a deformed Woods-Saxon potential.

Stimulation of GABAB receptors increases the expression of the proenkephalin gene in slice cultures of rat neocortex.

In rat neocortex the proenkephalin gene is expressed in GABAergic interneurons. Immunocytochemistry and in situ hybridisation show only a small number of cells in layers II to VI which express the gene. In organotypic slices of rat neocortex, the GABAA receptor inhibitor bicuculline methiodide enhances the expression of the gene in numerous cells. In the present study, we have investigated how GABA regulates the expression of the proenkephalin gene. The GABAA receptor antagonist bicuculline methiodide and the inhibitor of ligand-gated Cl- channels picrotoxin strongly enhanced the expression of the gene in numerous cells which were arranged in neocortical layers II/III and V/VI. Since bicuculline methiodide can also block Ca(++)-activated K+ channels, the possible involvement of such channels was tested. However, apamin which blocks only Ca(++)-activated K+ channels had no effect on the expression of the proenkephalin gene indicating that the effect of bicuculline methiodide was due to inhibition of GABAA receptors. In addition, the GABAB receptor agonist baclofen increased the neocortical expression of the proenkephalin gene mainly in cells located in layers V/VI of the neocortex. The effect of baclofen was inhibited by the GABAB receptor antagonists CGP35348 and CGP52432. Also muscimol, an agonist at GABAA receptors, enhanced the expression of the proenkephalin gene. This effect was blocked by CGP52432 confirming previous observations that muscimol can also stimulate GABAB receptors. Our results indicate that GABA can regulate the expression of the opioid peptide in neocortical neurons in a bidirectional manner. The expression is suppressed via GABAA and enhanced via GABAB receptors.

Stimulation of mouse cultured sympathetic neurons by uracil but not adenine nucleotides.

Cultured neurons from the paravertebral sympathetic chain of rats possess excitatory P2X as well as excitatory uracil nucleotide-sensitive P2Y receptors. Preliminary observations had indicated that the analogous neurons of mice lacked P2X receptors. This difference was now investigated. Thoracolumbar sympathetic neurons from one- to three-day-old mice were cultured for seven days. When the neurons were preincubated with [3H]noradrenaline and then superfused, ATP failed to cause any change in tritium outflow. UTP (3-300 microM) and UDP (30-100 microM), in contrast, caused marked increases, and so did nicotine (3-100 microM). The effect of UTP was not changed by suramin but abolished by tetrodotoxin and in the absence of calcium. The effect of nicotine was antagonized by hexamethonium and also abolished by tetrodotoxin and in the absence of calcium. Pre-exposure to UDP prevented the effect of UTP. In neurons studied by means of whole-cell patch-clamp techniques under current clamp, ATP lacked any effect. UTP (100 microM), UDP (100 microM) and nicotine (10 microM) caused depolarization accompanied by action potentials. Pre-exposure to UDP prevented the effect of UTP. In neurons studied under voltage clamp, ATP, UTP and UDP failed to cause any detectable current. Nicotine (10 microM), in contrast, elicited inward currents. Neither UTP nor UDP reduced the M-type potassium outward current. These results demonstrate a pronounced difference between cultured sympathetic neurons from the mouse and the rat paravertebral chain. Neurons from both species possess the nicotinic acetylcholine receptor. Neurons from both species also possess uracil nucleotide-sensitive P2Y receptors which, when activated, mediate depolarization, action potential firing and noradrenaline release; these effects are not due to inhibition of M-type potassium channels. Only the rat but not the mouse neurons, however, possess P2X receptors which, when activated, mediate cation entry, depolarization, action potential generation and transmitter release. The absence of functional P2X receptors makes the mouse neurons suitable for further study of the uracil nucleotide-sensitive P2Y receptors.

Alpha2-adrenoceptor-mediated inhibition of cultured sympathetic neurons: changes in alpha2A/D-adrenoceptor-deficient mice.

Alpha2-Adrenoceptor-mediated inhibition of [3H]noradrenaline release and alpha2-adrenoceptor-mediated inhibition of voltage-activated Ca2+ currents were compared in cultured thoracolumbar postganglionic sympathetic neurons from newborn wildtype (WT) mice and mice in which the alpha2A/D-adrenoceptor gene had been disrupted (alpha2A/DKO). In cultures prepared from WT mice and preincubated with [3H]noradrenaline, the alpha2-adrenoceptor agonist 5-bromo-6-(2-imidazolidinylidenamino)quinoxaline (UK 14,304) reduced the (autoinhibition-free) release of [3H]noradrenaline elicited by single electrical pulses or trains of 8 pulses at 100 Hz. The maximal inhibition by UK 14,304 amounted to 70%-85%. Its concentration-response curve was shifted to the right by phentolamine (0.3 microM) and, to a smaller extent, rauwolscine (0.3 microM). Pretreatment of the cultures with pertussis toxin abolished the effect of UK 14,304. Phentolamine and rauwolscine increased the (alpha2-autoinhibited) release of [3H]noradrenaline elicited by 18, 36 or 72 pulses at 3 Hz. In cultures from alpha2A/DKO mice, UK 14,304 failed to reduce the release of [3H]noradrenaline elicited by single pulses and phentolamine and rauwolscine failed to increase the release of [3H]noradrenaline elicited by 18-72 pulses at 3 Hz. In neurons from WT mice examined with the amphotericin B-perforated configuration of the patch clamp method, UK 14,304 reduced depolarisation-evoked Ca2+ currents. The inhibition was voltage-dependent as shown by a decline at strong depolarisation during ramp-like voltage commands and by an attenuation briefly after a conditioning depolarising pulse. The maximal inhibition by UK 14,304 was 39%. Its concentration-response curve was shifted to the right by phentolamine (0.3 microM) but not significantly changed by rauwolscine (0.3 microM) and prazosin (1 microM). Pretreatment with pertussis toxin abolished the effect of UK 14,304. In neurons from alpha2A/DKO mice, UK 14,304 also reduced depolarisation-evoked Ca2+ currents, but with a smaller maximal effect, namely 18% inhibition. Its concentration-response curve was shifted to the right by rauwolscine (0.3 microM) and prazosin (1 microM) but not significantly changed by phentolamine (0.3 microM). Pretreatment with pertussis toxin abolished the effect of UK 14,304 also in cultures from alpha2A/DKO mice. It is concluded that the only presynaptic alpha2-autoreceptors that detectably depress transmitter release from cultured thoracolumbar sympathetic neurons taken from newborn mice are alpha2A/D. In contrast, the soma-dendritic alpha2-autoreceptors that inhibit voltage-gated Ca2+ channels are both alpha2A/D and non-alpha2A/D (i.e. alpha2B or alpha2c). Both presynaptic alpha2A/D- and soma-dendritic alpha2A/D- and non-alpha2A/D-autoreceptors operate through pertussis toxin-sensitive G proteins in these neurons.

Neuronal P2X receptors: localisation and functional properties.

ATP is a co-transmitter in the central and peripheral nervous system. Extracellular ATP exerts its effects via ionotropic (P2X), as well as metabotropic receptors (P2Y). P2X receptors are involved in fast excitatory synaptic signalling by ATP, whereas the role of P2Y receptors in synaptic transmission is unclear. Seven different mammalian P2X receptor subunits (P2X1-7) have been cloned to date. This article gives an overview about the distribution of these P2X receptor subunits in the nervous system. A comparison is made between the pharmacological properties of recombinant receptors and natively occurring neuronal P2X receptors by means of electrophysiological methods. The subcellular distribution of, developmental influences on, and interspecies differences between P2X receptors are also considered. It is concluded that the properties of native P2X receptors are best explained by a heteromeric assembly of different P2X receptor subunits.

Regulation of somatodendritic GABAA receptor channels in rat hippocampal neurons: evidence for a role of the small GTPase Rac1.

The role of the cytoskeleton in the activity of GABA(A) receptors was investigated in cultured hippocampal neurons. Receptor currents were measured with the whole-cell patch-clamp technique during repetitive stimulation with 1 microm muscimol. After destruction of the microtubular system with nocodazol, muscimol-induced currents showed a rundown by 78%. A similar rundown was observed when actin fibers were destroyed with latrunculin B or C2 toxin of Clostridium botulinum. Because the small GTPases of the Rho family RhoA, Rac1, and Cdc42 are known to control the organization of actin fibers, we investigated their possible involvement. Inactivation of the GTPases with clostridial toxins, as well as intracellular application of recombinant Rho GTPases, indicated that active Rac1 was necessary for full GABA(A) receptor activity. Immunocytochemical labeling of the receptors showed that the disappearance of receptor clusters in the somatic membrane as induced by muscimol stimulation was enhanced by Rac1 inactivation. It is suggested that Rac1 participates in the regulation of GABA(A) receptor clustering and/or recycling.

Inhibition by adenosine A(2A) receptors of NMDA but not AMPA currents in rat neostriatal neurons.

Whole-cell patch clamp experiments were used to investigate the transduction mechanism of adenosine A(2A) receptors in modulating N-methyl-D-aspartate (NMDA)-induced currents in rat striatal brain slices. The A(2A) receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680) inhibited the NMDA, but not the (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) current in a subset of striatal neurons. Lucifer yellow-filled pipettes in combination with immunostaining of A(2A) receptors were used to identify CGS 21680-sensitive cells as typical medium spiny striatal neurons. Dibutyryl cyclic AMP and the protein kinase A activator Sp-cyclic AMPs, but not the protein kinase A inhibitors Rp-cyclic AMPS or PKI(14 - 24)amide abolished the inhibitory effect of CGS 21680. The phospholipase C inhibitor U-73122, but not the inactive structural analogue U-73343 also interfered with CGS 21680. The activation of protein kinase C by phorbol 12-myristate 13-acetate or the blockade of this enzyme by staurosporine did not alter the effect of CGS 21680. Heparin, an antagonist of inositol 1, 4,5-trisphosphate (InsP(3)) and a more efficient buffering of intracellular Ca(2+) by BAPTA instead of EGTA in the pipette solution, abolished the CGS 21680-induced inhibition. The calmodulin antagonist W-7 and cytochalasin B which enhances actin depolymerization also prevented the effect of CGS 21680; the calmodulin kinase II inhibitors CaM kinase II(281 - 309) and KN-93 but not the inactive structural analogue KN-92 were also effective. The calcineurin inhibitor deltamethrin did not interfere with CGS 21680. It is suggested that the transduction mechanism of A(2A) receptors to inhibit NMDA receptor channels is the phospholipase C/InsP(3)/calmodulin and calmodulin kinase II pathway. The adenylate cyclase/protein kinase A and phospholipase C/protein kinase C pathways do not appear to be involved.

M-type K+ currents in rat cultured thoracolumbar sympathetic neurones and their role in uracil nucleotide-evoked noradrenaline release.

Cultured sympathetic neurones are depolarized and release noradrenaline in response to extracellular ATP, UDP and UTP. We examined the possibility that, in neurones cultured from rat thoracolumbar sympathetic ganglia, inhibition of the M-type potassium current might underlie the effects of UDP and UTP. Reverse transcriptase-polymerase chain reaction indicated that the cultured cells contained mRNA for P2Y(2)-, P2Y(4)- and P2Y(6)-receptors as well as for the KCNQ2- and KCNQ3-subunits which have been suggested to assemble into M-channels. In cultures of neurones taken from newborn as well as from 10 day-old rats, oxotremorine, the M-channel blocker Ba(2+) and UDP all released previously stored [(3)H]-noradrenaline. The neurones possessed M-currents, the kinetic properties of which were similar in neurones from newborn and 9 - 12 day-old rats. UDP, UTP and ATP had no effect on M-currents in neurones prepared from newborn rats. Oxotremorine and Ba(2+) substantially inhibited the current. ATP also had no effect on the M-current in neurones prepared from 9 - 12 day-old rats. Oxotremorine and Ba(2+) again caused marked inhibition. In contrast to cultures from newborn animals, UDP and UTP attenuated the M-current in neurones from 9 - 12 day-old rats; however, the maximal inhibition was less than 30%. The results indicate that inhibition of the M-current is not involved in uracil nucleotide-induced transmitter release from rat cultured sympathetic neurones during early development. M-current inhibition may contribute to release at later stages, but only to a minor extent. The mechanism leading to noradrenaline release by UDP and UTP remains unknown.

Role of action potentials and calcium influx in ATP- and UDP-induced noradrenaline release from rat cultured sympathetic neurones.

Adenine and uracil nucleotides release noradrenaline from rat postganglionic sympathetic neurones by activation of P2X-receptors and distinct receptors for uracil nucleotides, respectively. The present study on cultured neurones of rat thoracolumbal paravertebral ganglia has analysed the involvement of action potentials and calcium influx in the nucleotide-induced transmitter release. ATP and UDP (100 microM each) caused a marked release of previously incorporated [3H]noradrenaline. The P2-receptor antagonists suramin (300 microM) and cibacron blue 3GA (3 microM) decreased the ATP-induced but not the UDP-induced release. The response to ATP was decreased by the sodium channel blocker tetrodotoxin (0.5 microM; by 47%), by the N-type calcium channel blocker omega-conotoxin GVIA (100 nM; by 35%), and by the alpha2-adrenoceptor agonist UK-14,304 (1 microM; by 45%); it was not changed by the potassium channel blocker tetraethylammonium (10 mM). The response to UDP was abolished by tetrodotoxin, greatly decreased by omega-conotoxin (by 78%), also abolished by UK-14,304, and increased by tetraethylammonium (by 410%). ATP (100 microM) caused a marked increase in intra-axonal free calcium as measured by fura-2 microfluorimetry. Withdrawal of extracellular calcium diminished the calcium response to ATP by 85%, and tetrodotoxin and omega-conotoxin diminished it by about 40%. As studied with the amphotericin B-perforated patch method, ATP (100 microM) induced a large depolarisation and action potential firing. UDP (100 microM) induced only a small depolarisation but it also elicited action potentials. UDP increased the excitability of the neurones. The results indicate that the ATP-induced release of noradrenaline depends on influx of calcium from the extracellular space. Calcium passes through two structures: voltage-gated channels and - probably - the P2X-receptor itself. Only that component of ATP-induced transmitter release which is triggered by opening of voltage-gated calcium channels is sensitive to modulation by alpha2-adrenoceptors. In contrast to ATP, the UDP-induced release of noradrenaline is entirely due to generation of action potentials followed by calcium influx through voltage-gated channels. All of the UDP-induced release is therefore sensitive to alpha2-adrenoceptor modulation.

Rundown of somatodendritic N-methyl-D-aspartate (NMDA) receptor channels in rat hippocampal neurones: evidence for a role of the small GTPase RhoA.

Actin filament (F-actin) depolymerization leads to the use-dependent rundown of N-methyl-D-aspartate (NMDA) receptor activity in rat hippocampal neurones. Depolymerization is promoted by Ca2+ which enters the cells via NMDA receptor channels. The ras homologue (Rho) GTPases (RhoA, Rac1 and Cdc42) promote actin polymerization and thus control the actin cytoskeleton. We have investigated, by means of the whole-cell patch clamp technique, whether the actin fibres which interact with NMDA receptors are controlled by Rho GTPases. In the presence of intracellular ATP which attenuates rundown, the C3 toxin from Clostridium (C.) botulinum was used to inactivate RhoA. Indeed, it enhanced the use-dependent rundown of NMDA-evoked inward currents to a level similar to that obtained in the absence of ATP. Lethal toxin from Clostridium sordellii which inactivates Rac1 and Cdc42 lacked this effect. We suggest that the function of somatodendritic NMDA receptor channels in rat hippocampal neurones can be modulated by RhoA via its action on F-actin.

Correlations of eigenvectors for non-Hermitian random-matrix models.

We establish a general relation between the diagonal correlator of eigenvectors and the spectral Green's function for non-Hermitian random-matrix models in the large-N limit. We apply this result to a number of non-Hermitian random-matrix models and show that the outcome is in good agreement with numerical results.

Inhibition of GABAergic inhibitory postsynaptic currents by cannabinoids in rat corpus striatum.

Electrophysiological consequences of activation of cannabinoid receptors have been mostly investigated on neuronal cell lines and on cells transfected with cannabinoid receptors. The aim of the present experiments was to study cannabinoid effects on identified neurons in situ. Electrically-evoked postsynaptic currents and voltage-dependent calcium currents were investigated in the principal neurons of the corpus striatum, the medium spiny neurons, with the patch-clamp method for brain slices. These neurons were chosen because they produce messenger RNA for cannabinoid receptors and because the density of cannabinoid binding sites in the striatum is high. Activation of muscarinic receptors by carbachol (10(-5) M) reduced inhibitory postsynaptic current amplitude by 67%. The synthetic cannabinoid receptor agonist R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4- benzoxazin-yl]-(1-naphtalenyl)methanone (WIN55212-2; 10(-8) to 10(-5) M) dose-dependently reduced striatal inhibitory postsynaptic currents; the maximum effect, inhibition by 52%, was observed at 10(-6) M. Another cannabinoid agonist, (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydr oxypropyl)cyclohexanol (CP55940; 10(-6) M), also reduced inhibitory postsynaptic currents, by 50%. The CB1 cannnabinoid receptor antagonist N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)4-methyl-3-pyra zolecarboxamide (SR141716A; 10(-6) M) had no effect when given alone but abolished the effect of WIN55212-2 (10(-6) M). WIN55212-2 (10(-6) M) did not change the current evoked by the GABA(A)-receptor agonist muscimol (10(-6) M). Activation of muscarinic receptors by carbachol (10(-5) M) inhibited voltage-dependent calcium currents by 21%, but the cannabinoid receptor agonist WIN55212-2 (10(-6) M) was without effect. The results show that activation of CB1 cannabinoid receptors reduces GABAergic inhibitory postsynaptic currents in medium spiny neurons of the corpus striatum: the likely mechanism is presynaptic inhibition of GABA release from terminals of recurrent axons of the medium spiny neurons themselves.

Adenosine A2A receptors inhibit the conductance of NMDA receptor channels in rat neostriatal neurons.

Whole-cell patch clamp experiments were carried out in rat striatal brain slices. In a subset of striatal neurons (70-80%), NMDA-induced inward currents were inhibited by the adenosine A2A receptor selective agonist CGS 21680. The non-selective adenosine receptor antagonist 8-(p-sulphophenyl)-theophylline and the A2A receptor selective antagonist 8-(3-chlorostyryl)caffeine abolished the inhibitory action of CGS 21680. Intracellular GDP-beta-S, which is known to prevent G protein-mediated reactions, also eliminated the effect of CGS 21680. Extracellular dibutyryl cAMP, a membrane permeable analogue of cAMP, and intracellular Sp-cAMPS, an activator of cAMP-dependent protein kinases (PKA), both abolished the CGS 21680-induced inhibition. By contrast, Rp-cAMPS and PKI 14-24 amide, two inhibitors of PKA had no effect. Intracellular U-73122 (a phospholipase C inhibitor) and heparin (an inositoltriphosphate antagonist) prevented the effect of CGS 21680. Finally, a more efficient buffering of intracellular Ca2+ by a substitution of EGTA (11 mM) by BAPTA (5.5 mM) acted like U-73122 or heparin. Hence, A2A receptors appear to negatively modulate NMDA receptor channel conductance via the phospholipase C/inositoltriphosphate/Ca2+ pathway rather than the adenylate cyclase/PKA pathway.

Effect of adenosine and some of its structural analogues on the conductance of NMDA receptor channels in a subset of rat neostriatal neurones.

1. In order to investigate the modulatory effects of adenosine on excitatory amino acid projections onto striatal medium spiny neurons, whole-cell patch clamp experiments were carried out in rat brain slices. The effects of various agonists for P1 (adenosine) and P2 (ATP) purinoceptors and their antagonists were investigated. The A2A receptor agonist 2-p-(2-carboxyethyl)phenythylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680; 0.1 microM), the A1 receptor agonist 2-chloro-N6-cyclcopentyladenosine (CCPA; 10 microM) and the non-selective P1 purinoceptor antagonist 8-(p sulphophenyl)-theophylline (8-SPT; 100 microM) did not alter the resting membrane potential, the threshold current necessary to elicit an action potential, the amplitude of spikes, their rise time, the amplitude of the afterhyperpolarization (AHP) and the time to peak of the AHP. 2. N-methyl-D-aspartate (NMDA; 1-1000 microM) caused a concentration-dependent inward current which was larger in the absence than in the presence of Mg2+ (1.3 mM). In a subset of striatal neurones, the current response to NMDA (10 microM) and the accompanying increase in conductance were both inhibited by CGS 21680 (0.01-1 microM). The effect of CGS 21680 (0.1 microM) persisted in the presence of tetrodotoxin (0.5 microM) or in a Ca(2+)-free medium, under conditions when synaptically mediated influences may be negligible. 3. The A3 receptor agonist N6-2-(4-aminophenyl)ethyladenosine (APNEA; 0.1-10 microM) also diminished the effect of NMDA (10 microM), while the A1 receptor agonists CCPA (0.1-10 microM) and (2S)-N6-[2-endonorbornyl] adenosine [S(-)-ENBA; 10 microM] as well as the endogenous, non-selective P1 purinoceptor agonist adenosine (100 microM) were inactive. The endogenous non-selective P2 purinoceptor agonist ATP (1000 microM) also failed to alter the current response to NMDA (10 microM). Adenosine (100 microM), but not ATP (1000 microM) became inhibitory after blockade of nucleoside uptake by S[4-nitrobenzyl)-6-thioguanosine (NBTG; 30 microM). 4. 8-(p-Sulphophenyl)-theophylline (8-SPT; 100 microM), as well as the A2A receptor antagonist 8-(3-chlorostyryl) caffeine (CSC; 1 microM) and the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) at 0.03, but not 0.003 microM abolished the inhibitory action of CGS 21,680 (0.1 microM). None of these compounds altered the effect of NMDA (10 microM) by itself. DPCPX (0.03 microM) prevented the inhibition of APNEA (10 microM). 5. There was no effect of CGS 21,680 (0.1 microM), when guanosine 5'-O-(3-thiodiphosphate (GDP-beta-S; 300 microM) was included in the pipette solution in order to block G protein-mediated reactions. 6. In conclusion, adenosine receptors, probably of the A2A-subtype, inhibit the conductance of NMDA receptor channels in a subset of medium spiny neurones of the rat striatum by a transduction mechanism which involves a G protein.