Functional expression of ionotropic purinergic receptors on mouse taste bud cells

Neurotransmitter receptors on taste bud cells (TBCs) and taste nerve fibres are likely to contribute to taste transduction by mediating the interaction among TBCs and that between TBCs and taste nerve fibres. We investigated the functional expression of P2 receptor subtypes on TBCs of mouse fungiform papillae. Electrophysiological studies showed that 100 μ m ATP applied to their basolateral membranes either depolarized or hyperpolarized a few cells per taste bud. Ca²⁺ imaging showed that similarly applied 1 μ m ATP, 30 μ m BzATP (a P2X₇ agonist), or 1 μ m 2MeSATP (a P2Y₁ and P2Y₁₁ agonist) increased intracellular Ca²⁺ concentration, but 100 μ m UTP (a P2Y₂ and P2Y₄ agonist) and α,β-meATP (a P2X agonist except for P2X₂, P2X₄ and P2X₇) did not. RT-PCR suggested the expression of P2X₂, P2X₄, P2X₇, P2Y₁, P2Y₁₃ and P2Y₁₄ among the seven P2X subtypes and seven P2Y subtypes examined. Immunohistostaining confirmed the expression of P2X₂. The exposure of the basolateral membranes to 3 m m ATP for 30 min caused the uptake of Lucifer Yellow CH in a few TBCs per taste bud. This was antagonized by 100 μ m PPADS (a non-selective P2 blocker) and 1 μ m KN-62 (a P2X₇ blocker). These results showed for the first time the functional expression of P2X₂ and P2X₇ on TBCs. The roles of P2 receptor subtypes in the taste transduction, and the renewal of TBCs, are discussed.     

Dehydroepiandrosterone Potentiates Native Ionotropic ATP Receptors Containing the P2X2 Subunit in Rat Sensory Neurones

We have studied the modulatory effect of dehydroepiandrosterone (DHEA), the most abundant neurosteroid produced by glial cells and neurones, on membrane currents induced by the activation of ionotropic ATP (P2X) receptors in neonatal rat dorsal root ganglion neurones. ATP (1 μ m ) induced three types of currents/responses termed F (fast and transient), S (slowly desensitizing) and M (mixed, sum of F- and S-type responses). DHEA (10 n m to 100 μ m ) concentration-dependently increased the amplitude of plateau-like currents of S- and M-type responses evoked by submaximal (1 μ m ) but not saturating (100 μ m or 1 mM) concentrations of ATP. αβ-Methylene ATP (αβme-ATP, 5 μ m ) also evoked F-, S- and M-type responses, the plateau phases of which were potentiated by lowering external pH (6.3) and by ivermectin (IVM, 3 μ m ), indicating the presence heteromeric P2X₂-containing receptors and possibly of functional native P2X_4/6 receptors. There was a strict correlation between the potentiating effects of low pH and DHEA on αβme-ATP responses but not between that of IVM and DHEA, suggesting that DHEA selectively modulated P2X₂-containing receptors. DHEA also potentiated putative homomeric P2X₂ receptor responses recorded in the continuous presence of 1 μ m 2'-(or 3')- O -(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP). Our results constitute the first demonstration of a fast potentiation of P2X receptors by a neurosteroid and suggest that DHEA could be an endogenous modulator of P2X₂-containing receptors thereby contributing to the facilitation of the detection and/or the transmission of nociceptive messages, particularly under conditions of inflammatory pain where the P2X receptor signalling pathway appears to be upregulated.     

Defining electrical communication in skeletal muscle resistance arteries: a computational approach

Lumbar splanchnic (LSN) and sacral pelvic (PN) nerves convey different mechanosensory information from the colon to the spinal cord. Here we determined whether these pathways also differ in their chemosensitivity and receptor expression. Using an in vitro mouse colon preparation, individual primary afferents were tested with selective P2X and transient receptor potential vanilloid receptor 1 (TRPV1) receptor ligands. Afferent cell bodies in thoracolumbar and lumbosacral dorsal root ganglia (DRG) were retrogradely labelled from the colon and analysed for P2X₃- and TRPV1-like immunoreactivity (LI). Forty per cent of LSN afferents responded to α,β-methylene adenosine 5'-triphosphate (α,β-meATP; 1 m m ), an effect that was concentration dependent and reversed by the P2X antagonist pyridoxyl5-phosphate 6-azophenyl-2',4'-disulphonic acid (PPADS) (100 μ m ). Significantly fewer PN afferents (7%) responded to α,β-meATP. Correspondingly, 36% of colonic thoracolumbar DRG neurones exhibited P2X₃-LI compared with only 19% of colonic lumbosacral neurones. Capsaicin (3 μ m ) excited 61% of LSN afferents and 47% of PN afferents; 82% of thoracolumbar and 50% of lumbosacral colonic DRG neurones displayed TRPV1-LI. Mechanically insensitive afferents were recruited by α,β-meATP or capsaicin, and were almost exclusive to the LSN. Capsaicin-responsive LSN afferents displayed marked mechanical desensitization after responding to capsaicin, which did not occur in capsaicin-responsive PN afferents. Therefore, colonic LSN and PN pathways differ in their chemosensitivity to known noxious stimuli and their corresponding receptor expression. As these pathways relay information that may relate to symptoms in functional gastrointestinal disease, these results may have implications for the efficacy of therapies targeting receptor modulation.     

Mechanisms of acid-induced activation of airway afferent nerve fibres in guinea-pig

It has been proposed that extracellular ATP may be involved in visceral mechanosensory transduction by activating ligand-gated ion channels (P2X receptors). In this study, we have investigated the effects of the P2X₃ agonist α,β-methylene ATP (α,β-meATP) and antagonist 2',3'- O -trinitrophenyl-ATP (TNP-ATP) on pelvic afferents innervating the urinary bladder using an in vitro mouse bladder-pelvic nerve preparation. Intravesical application of α,β-meATP (0.03-1 m m ) increased multifibre discharges in a concentration-dependent manner. The agonist potentiated, whereas TNP-ATP (0.03 m m ) attenuated, the multifibre responses to bladder distensions. Single-unit analysis revealed that both high threshold (HT) fibres (> 15 mmHg; known to be associated with nociception) and low threshold (LT) fibres (< 15 mmHg; probably associated with non-nociceptive events) could be induced to discharge by intravesical α,β-meATP (1 m m , 0.1 ml). The response of the vast majority (21/22, 95.5 %) of HT fibres to bladder distensions was enhanced with a significantly reduced threshold and an increased peak response after exposure to the agonist. On the other hand, 59.7 % (46/77) of LT fibres showed a greater peak and a slightly reduced threshold for response to bladder distension in the presence of α,β-meATP. An additional 11 'silent' fibres became mechanosensitive after exposure to α,β-meATP. TNP-ATP (0.03 m m ) did not affect the threshold of LT fibres, but it reduced the peak response of some (22/51, 43.1 %) LT fibres. Conversely, the antagonist resulted in a markedly elevated threshold and reduced peak activity in the majority (13/16, 81.3 %) of HT fibres. The results support the view that P2X₃ receptor-mediated mechanisms contribute to both nociceptive and non-nociceptive (physiological) mechanosensory transduction in the urinary bladder.     

The role of histidine residues in modulation of the rat P2X2 purinoceptor by zinc and pH

P2X₂ receptor currents are potentiated by acidic pH and zinc. To identify residues necessary for proton and zinc modulation, alanines were singly substituted for each of the nine histidines in the extracellular domain of the rat P2X₂ receptor. Wild-type and mutant receptors were expressed in Xenopus oocytes and analysed with two-electrode voltage clamp. All mutations caused less than a 2-fold change in the EC₅₀ of the ATP concentration-response relation. Decreasing the extracellular pH from 7.5 to 6.5 potentiated the responses to 10 μ m ATP of wild-type P2X₂ and eight mutant receptors more than 4-fold, but the response of the mutant receptor H319A was potentiated only 1.4-fold. The H319A mutation greatly attenuated the maximal potentiation that could be produced by a drop in pH, shifted the p K _a (-log of dissociation constant) of the potentiation to a more basic pH as compared with P2X₂ and revealed a substantial pH-dependent decrease in the maximum response with a p K _a near 6.0. Substituting a lysine for H319 reduced the EC₅₀ for ATP 40-fold. Zinc (20 μ m ) potentiated the responses to 10 μ m ATP of wild-type P2X₂ and seven histidine mutants by ∼8-fold but had virtually no effect on the responses of two mutants, H120A and H213A. Neither H120A nor H213A removed the voltage-independent inhibition caused by high concentrations of zinc. The observation that different mutations selectively eliminated pH or zinc potentiation implies that there are two independent sites of action, even though the mechanisms of pH and zinc potentiation appear similar.     

Functional roles of presynaptic GABAA receptors on glycinergic nerve terminals in the rat spinal cord

GABA_A receptor-mediated presynaptic depolarization is believed to induce presynaptic inhibition of excitatory synaptic transmission. We report here the functional roles of presynaptic GABA_A receptors in glycinergic transmission of the rat spinal cord. In mechanically dissociated rat sacral dorsal commissural nucleus (SDCN) neurons attached with native glycinergic and GABAergic nerve terminals, glycinergic spontaneous inhibitory postsynaptic currents (sIPSCs) were isolated from a mixture of both glycinergic and GABAergic sIPSCs by perfusing the SDCN nerve cell body with ATP-free internal solution. Under such experimental conditions, exogenously applied muscimol (0.5 μM) depolarized glycinergic presynaptic nerve terminals and significantly increased glycinergic sIPSC frequency to 542.7 ± 47.3 % of the control without affecting the mean current amplitude. The facilitatory effect of muscimol on sIPSC frequency was completely blocked by bicuculline (10 μM) or SR95531 (10 μM), selective GABA_A receptor antagonists. This muscimol-induced presynaptic depolarization was due to a higher intraterminal Cl⁻ concentration, which is maintained by a bumetanide-sensitive Na-K-Cl cotransporter. On the contrary, when electrically evoked, this muscimol-induced presynaptic depolarization was found to decrease the action potential-dependent glycine release evoked by focal stimulation of a single terminal. The results suggest that GABA_A receptor-mediated presynaptic depolarization has two functional roles: (1) presynaptic inhibition of action potential-driven glycinergic transmission, and (2) presynaptic facilitation of spontaneous glycinergic transmission.     

Activation of GABAA receptors containing the α4 subunit by GABA and pentobarbital

The activation properties of GABA_A receptors containing α4β2γ2 and α4β2δ subunits were examined in the presence of GABA or pentobarbital. The receptors were expressed transiently in HEK 293 cells, and the electrophysiological experiments were carried out using cell-attached single-channel patch clamp or whole-cell macroscopic recordings. The data show that GABA is a stronger activator of α4β2γ2 receptors than α4β2δ receptors. Single-channel clusters were recorded from α4β2γ2 receptors in the presence of 10–5000 μ m GABA. The maximal intracluster open probability was 0.35, with a half-maximal response elicited by 32 μ m GABA. Simultaneous kinetic analysis of single-channel currents obtained at various GABA concentrations yields a channel opening rate constant of 250 s⁻¹, and a K _D of 20 μ m . In contrast, only isolated openings were observed in the presence of GABA for the α4β2δ receptor. Pentobarbital was a strong activator of both α4β2γ2 and α4β2δ receptors. The maximal cluster open probability, recorded in the presence of 100 μ m pentobarbital, was 0.7. At higher pentobarbital concentrations, the cluster open probability was reduced, probably due to channel block. The results from single-channel experiments were confirmed by macroscopic recordings from HEK cells in the presence of GABA or pentobarbital.     

Protein kinase C mediates up-regulation of tetrodotoxin-resistant, persistent Na+ current in rat and mouse sensory neurones

The tetrodotoxin-resistant (TTX-r) persistent Na⁺ current, attributed to Na_V1.9, was recorded in small (< 25 μm apparent diameter) dorsal root ganglion (DRG) neurones cultured from P21 rats and from adult wild-type and Na_V1.8 null mice. In conventional whole-cell recordings intracellular GTP-γ-S caused current up-regulation, an effect inhibited by the PKC pseudosubstrate inhibitor, PKC19–36. The current amplitude was also up-regulated by 25 μ m intracellular 1-oleoyl-2-acetyl-sn-glycerol (OAG) consistent with PKC involvement. In perforated-patch recordings, phorbol 12-myristate 13-acetate (PMA) up-regulated the current, whereas membrane-permeant activators of protein kinase A (PKA) were without effect. PGE₂ did not acutely up-regulate the current. Conversely, both PGE₂ and PKA activation up-regulated the major TTX-r Na⁺ current, Na_V1.8. Extracellular ATP up-regulated the persistent current with an average apparent K _d near 13 μ m , possibly consistent with P2Y receptor activation. Numerical simulation of the up-regulation qualitatively reproduced changes in sensory neurone firing properties. The activation of PKC appears to be a necessary step in the GTP-dependent up-regulation of persistent Na⁺ current.     

Excitatory effects of serotonin on rat striatal cholinergic interneurones

We investigated the effects of 5-hydroxytryptamine (5-HT, serotonin) in striatal cholinergic interneurones with gramicidin-perforated whole-cell patch recordings. Bath-application of serotonin (30 μ m ) significantly and reversibly increased the spontaneous firing rate of 37/45 cholinergic interneurones tested. On average, in the presence of serotonin, firing rate was 273 ± 193% of control. Selective agonists of 5-HT_1A, 5-HT₃, 5-HT₄ and 5-HT₇ receptors did not affect cholinergic interneurone firing, while the 5-HT₂ receptor agonist α-methyl-5-HT (30 μ m ) mimicked the excitatory effects of serotonin. Consistently, the 5-HT₂ receptor antagonist ketanserin (10 μ m ) fully blocked the excitatory effects of serotonin. Two prominent after-hyperpolarizations (AHPs), one of medium duration that was apamin-sensitive and followed individual spikes, and one that was slower and followed trains of spikes, were both strongly and reversibly reduced by serotonin; these effects were fully blocked by ketanserin. Conversely, the depolarizing sags observed during negative current injections and mediated by hyperpolarization-activated cationic currents were not affected. In the presence of apamin and tetrodotoxin, the slow AHP was strongly reduced by 5-HT, and fully abolished by the calcium channel blocker nickel. These results show that 5-HT exerts a powerful excitatory control on cholinergic interneurones via 5-HT₂ receptors, by suppressing the AHPs associated with two distinct calcium-activated potassium currents.     

Immunohistochemical identification of cells expressing ATP-gated cation channels (P2X receptors) in the adult rat thyroid

We carried out immunohistochemistry and western blotting of fresh frozen sections and crude extracts from adult rat thyroids. The histochemical and immunoblotting studies were performed with P2X receptor antibodies from 2 different sources. P2X-immunopositive cells were identified by fluorescence double labelling and confocal microscopy. Results of the western blotting experiments showed double bands of approximately 70 kDa and 140 kDa for all 7 P2X receptor subtypes with both sets of antibodies. Histochemical stains with antibodies from both sources also gave essentially identical results. P2X₁, P2X₂ and P2X₆ receptors were detected exclusively in vascular smooth muscle; P2X₅ and P2X₇ receptors were also present on vascular smooth muscle. Endothelial cells stained for P2X₃, P2X₄ and P2X₇ receptors. Thyroid follicular cells displayed immunoreactivity for P2X₃, P2X₄ and P2X₅ receptors. No immunostaining for P2X receptors was observed on C-cells. Possible roles for the broad expression of P2X receptor subtypes in the rat thyroid are discussed.     

Hypoxia potentiates exocytosis and Ca2+ channels in PC12 cells via increased amyloid β peptide formation and reactive oxygen species generation

Exposure of PC12 cells to chronic hypoxia (CH; 10 % O₂, 24 h) augments catecholamine secretion via formation of a Cd²⁺-resistant Ca²⁺ influx pathway, and up-regulates native L-type Ca²⁺ channels. These effects are mimicked by exposure of cells to Alzheimer's disease-associated amyloid β peptides (AβPs). Since pathological effects of AβPs have been associated with increased levels of reactive oxygen species (ROS), the involvement of ROS in hypoxia-mediated up-regulation of exocytosis and Ca²⁺ channel activity was examined. Both melatonin and ascorbic acid (two structurally unrelated antioxidants) fully blocked the enhancement of catecholamine secretion caused by CH (as determined amperometrically). Enhanced immunofluorescence, observed in chronically hypoxic cells using a primary monoclonal antibody raised against the N-terminus of AβP, was also suppressed by melatonin. Ascorbic acid, melatonin and ebselen (an additional antioxidant) also fully prevented augmentation of whole-cell Ca²⁺ currents caused by CH (as monitored using whole-cell patch-clamp recordings). Exposure of normoxic cells to H₂O₂ (40 μM, 24 h), like hypoxia, caused Ca²⁺ channel up-regulation. Importantly, AβP formation appeared to be an absolute requirement for the effects of hypoxia, since the ability of CH to augment exocytosis and Ca²⁺ channel activity was blocked by two novel inhibitors of γ secretase, an enzyme complex required for AβP formation. Our results indicate that the effects of hypoxia require ROS generation from AβPs, and suggest that elevated levels of ROS mediate hypoxic and AβP-mediated pathological remodelling of Ca²⁺ homeostasis.     

Dynamic regulation of glycinergic input to spinal dorsal horn neurones by muscarinic receptor subtypes in rats

Activation of spinal muscarinic acetylcholine receptors (mAChRs) inhibits nociception. However, the cellular mechanisms of this action are not fully known. In this study, we determined the role of mAChR subtypes in regulation of synaptic glycine release in the spinal cord. Whole-cell voltage-clamp recordings were performed on lamina II neurones in the rat spinal cord slices. The mAChR agonist oxotremorine-M significantly increased the frequency of glycinergic sIPSCs but not mIPSCs. Surprisingly, the effect of oxotremorine-M on sIPSCs was largely attenuated at a higher concentration. On the other hand, 1–10 μ m oxotremorine-M dose-dependently increased the frequency of sIPSCs in rats pretreated with intrathecal pertussis toxin. Furthermore, oxotremorine-M also dose-dependently increased the frequency of sIPSCs in the presence of himbacine (an M₂/M₄ mAChR antagonist) or AF-DX116 (an M₂ mAChR antagonist). The M₃ mAChR antagonist 4-DAMP abolished the stimulatory effect of oxotremorine-M on sIPSCs. Interestingly, the GABA_B receptor antagonist CGP55845 potentiated the stimulatory effect of oxotremorine-M on sIPSCs. In the presence of CGP55845, both himbacine and AF-DX116 similarly reduced the potentiating effect of oxotremorine-M on sIPSCs. Collectively, these data suggest that the M₃ subtype is present on the somatodendritic site of glycinergic neurones and is mainly responsible for muscarinic potentiation of glycinergic input to spinal dorsal horn neurones. Concurrent stimulation of mAChRs on adjacent GABAergic interneurones attenuates synaptic glycine release through presynaptic GABA_B receptors on glycinergic interneurones. This study illustrates a complex dynamic interaction between GABAergic and glycinergic synapses in the spinal cord dorsal horn.     

Effect of protease-activated receptor 2 activation on single TRPV1 channel activities in rat vagal pulmonary sensory neurons

Protease-activated receptor 2 (PAR₂) is involved in airway inflammation and airway hyperresponsiveness; both are the prominent features of asthma. Transient receptor potential vanilloid receptor 1 (TRPV1) is expressed in pulmonary sensory nerves, functions as a thermal and chemical transducer and contributes to neurogenic inflammation. Using cell-attached single-channel recordings we investigated the effect of PAR₂ activation on single TRPV1 channel activities in isolated pulmonary sensory neurons. Our immunohistochemical study demonstrated the expression of PAR₂ in rat vagal pulmonary sensory neurons. Our patch-clamp study further showed that intracellular application of capsaicin (0.75 μ m ) induced single-channel current that exhibited outward rectification in these neurons. The probability of the channel being open ( P _o) was significantly increased after the cells were pretreated with PAR₂-activating peptide (100 μ m , 2 min). Pretreatment with trypsin (0.1 μ m , 2 min) also increased the single-channel P _o, and the effect was completely inhibited by soybean trypsin inhibitor (0.5 μ m , 3 min). In addition, the effect of PAR₂ activation was abolished by either U73122 (1 μ m , 4 min), a phospholipase C inhibitor, or chelerythrine (10 μ m , 4 min), a protein kinase C inhibitor. In conclusion, our data demonstrated that activation of PAR₂ upregulated single-channel activities of TRPV1 and that the effect was mediated through the protein kinase C-dependent transduction pathway.     

Effects of 17β-oestradiol on rat detrusor smooth muscle contractility

The aim of this study was to investigate the effect of 17β-oestradiol (E₂) on detrusor smooth muscle contractility and its possible neuroprotective role against ischaemic-like condition, which could arise during overactive bladder disease. The effect of E₂ was investigated on rat detrusor muscle strips stimulated with carbachol, KCl and electrically, in the absence or presence of a selective oestrogen receptor antagonist (ICI 182,780) and, by using confocal Ca²⁺ imaging technique, measuring the amplitude (Δ F / F ₀) and the frequency of spontaneous whole cell Ca²⁺ flashes. Moreover, the effect of 1 and 2 h of anoxia–glucopenia and reperfusion (A-G/R), in the absence or presence of the hormone, was evaluated in rat detrusor strips perfused with Krebs solution which underwent electrical field stimulation to stimulate intrinsic nerves; the amplitude and the frequency of Ca²⁺ flashes were also measured. 17β-Oestradiol exhibited antispasmogenic activity assessed on detrusor strips depolarized with 60 m m KCl at two different Ca²⁺ concentrations. 17β-Oestradiol at the highest concentration tested (30 μ m ) significantly decreased detrusor contractions induced by all the stimuli applied. In addition, the amplitude and the frequency of spontaneous Ca²⁺ flashes were significantly decreased in the presence of E₂ (10 and 30 μ m ) compared with control detrusor strips. In strips subjected to A-G/R, a significant increase in the amplitude of both spontaneous and evoked flashes was observed. 17β-Oestradiol was found to increase the recovery of detrusor strips subjected to A-G/R. The ability of E₂ to suppress contraction in control conditions may explain its ability to aid recovery following A-G/R.     

Ca2+-desensitizing hypoxic vasorelaxation: pivotal role for the myosin binding subunit of myosin phosphatase (MYPT1) in porcine coronary artery

Acute hypoxia dilates most systemic arteries leading to increased tissue perfusion. We showed that at high stimulus conditions, porcine coronary artery was relaxed by hypoxia without a change in [Ca²⁺]_i. This 'Ca²⁺-desensitizing hypoxic relaxation' was validated in permeabilized porcine coronary artery smooth muscle (PCASM) in which hypoxia decreased force and myosin regulatory light chain phosphorylation (p-MRLC) despite fixed [Ca²⁺]. Rho kinase-dependent phosphorylation of MYPT1 (p-MYPT1) is associated with decreased MRLC phosphatase (MLCP) activity, and increased Ca²⁺ sensitivity of both p-MRLC and force. We tested the hypothesis that hypoxia induces Ca²⁺-desensitizing hypoxic relaxation via dephosphorylation of p-MYPT1, consequently increasing MLCP activity and thus decreasing p-MRLC. α-Toxin-permeabilized PCASM pretreated with ATPγS did not relax in response to hypoxia. Moreover, when MRLC but not MYPT1 was protected from ATPγS thiophosphorylation by the MRLC kinase inhibitor ML7 (300 μ m ), hypoxia remained ineffective. In contrast, hypoxic relaxation was preserved with further addition of the Rho kinase inhibitor Y27632 (1 μ m ), to attenuate thiophosphorylation of MYPT1. Importantly, measurements of p-MRLC, and p-MYPT1 at T696 and T853 (human sequence) paralleled that of force. We conclude that Ca²⁺-desensitizing hypoxic relaxation requires dephosphorylation of p-MYPT1. Moreover, no kinases, other then those inhibited by ML7 and Y27632, nor their associated phosphoproteins can be involved in Ca²⁺-desensitizing hypoxic relaxation.     

Cytokines and PGE2 Modulate the Phagocytic Function of the β-Glucan Receptor in Macrophages

Under serum-free conditions the β-glucan receptor of mouse macrophages mediates phagocytosis of β-l,3-D-glucan-coated microbeads (diameter 2 μm). IFN-γ increases the phagocytic function of the β-glucan receptor in a dose-dependent manner, giving the plateau level at 100 U/ml. Maximum activity appears 9 h after addition of IFN-γ to the cells. The effect disappears within 24 h. The effect of IFN-γ may be a result of augmented receptor synthesis since treatment with cycloheximide reduces the phagocytosis.
IL-1 also increases the phagocytic function of the β-glucan receptor giving a dose-dependent response and with the plateau level reached at 10 U/ml. Maximum activity is found 4 h after addition of IL-I to macrophages. The effect disappears within 24 h. TNF does not alter the phagocytic function of the β-glucan receptor, but TNF together with IL-1 prolongs the effect of IL-1.
PGE₂ reduces the phagocytic function of the β-glucan receptor. Maximum reduction is achieved with 8 ng/ml. Time-course studies show the lowest phagocytic activity 9 h after addition of PGE₂ to the cells.     

Functional glycine receptor maturation in the absence of glycinergic input in dopaminergic neurones of the rat substantia nigra

The postnatal maturation pattern of glycine receptor channels (GlyRs) expressed by dopaminergic (DA) neurones of the rat substantia nigra pars compacta (SNc) was investigated using single-channel and whole-cell patch-clamp recordings in brain slices from rats aged 7–21 postnatal days (P). In neonatal rats (P7-P10), GlyRs exhibited a main conductance state of 100–110 pS with a mean open time of 16 ms. In juvenile rats (P19-P22), both the GlyR main conductance state (46-55 pS) and the mean open time (6.8 ms) were decreased. In neonatal rats, application of 30 μ m picrotoxin, which is known to block homomeric GlyRs, strongly reduced glycine-evoked responses, while it was much less effective in juvenile rats. These results suggest that these GlyRs correspond functionally to α₂ homomeric GlyRs in neonatal rats and α₁/β heteromeric GlyRs in juvenile rats. A drastic but transient decrease in the glycine responsiveness of DA neurones occurred around P17 concomitant to the functional switch from the homomeric state to the heteromeric state. This age corresponds to a maturation phase for DA neurones. The application of 1 μ m gabazine blocked spontaneous or evoked inhibitory synaptic current, while the addition of 1 μ m strychnine had no effect, suggesting a lack of functional glycinergic synapses on DA neurones. Although it has been proposed that taurine is co-released with GABA at GABAergic synapses on DA neurones, in the present study the stimulation of GABAergic fibres failed to activate GlyRs. Blockade of taurine transporters and applications of high K⁺ and hyposmotic solutions were also unable to induce any strychnine-sensitive current. We conclude that functional maturation of GlyRs can occur in the absence of any detectable GlyR activation in DA neurones of the SNc.     

Caffeine inhibition of ionotropic glycine receptors

We found that caffeine is a structural analogue of strychnine and a competitive antagonist at ionotropic glycine receptors (GlyRs). Docking simulations indicate that caffeine and strychnine may bind to similar sites at the GlyR. The R131A GlyR mutation, which reduces strychnine antagonism without suppressing activation by glycine, also reduces caffeine antagonism. GlyR subtypes have differing caffeine sensitivity. Tested against the EC₅₀ of each GlyR subtype, the order of caffeine potency (IC₅₀) is: α2β (248 ± 32 μ m ) ≈α3β (255 ± 16 μ m ) > α4β (517 ± 50 μ m ) > α1β(837 ± 132 μ m ). However, because the α3β GlyR is more than 3-fold less sensitive to glycine than any of the other GlyR subtypes, this receptor is most effectively blocked by caffeine. The glycine dose–response curves and the effects of caffeine indicate that amphibian retinal ganglion cells do not express a plethora of GlyR subtypes and are dominated by the α1β GlyR. Comparing the effects of caffeine on glycinergic spontaneous and evoked IPSCs indicates that evoked release elevates the glycine concentration at some synapses whereas summation elicits evoked IPSCs at other synapses. Caffeine serves to identify the pharmacophore of strychnine and produces near-complete inhibition of glycine receptors at concentrations commonly employed to stimulate ryanodine receptors.     

P2X3 receptors and peripheral pain mechanisms

ATP released from damaged or inflamed tissues can act at P2X receptors expressed on primary afferent neurones. The resulting depolarization can initiate action potentials that are interpreted centrally as pain. P2X₃ subunits are found in a subset of small-diameter, primary afferent neurones, some of which are also sensitive to capsaicin. They can form homo-oligomeric channels, or they can assemble with P2X₂ subunits into hetero-oligomers. Studies with antagonists selective for P2X₃-containing receptors, experiments with antisense oligonucleotides to reduce P2X₃ subunit levels, and behavioural testing of P2X₃ knock-out mice, all suggest a role for the P2X_2/3 receptor in the signalling of chronic inflammatory pain and some features of neuropathic pain. The availability of such tools and experimental approaches promises to accelerate our understanding of the other physiological roles for P2X receptors on primary afferent neurones.