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.     

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.     

P2X receptor subtype-specific modulation of excitatory and inhibitory synaptic inputs in the rat brainstem

The role of P2 receptors in synaptic transmission to the rat medial nucleus of the trapezoid body (MNTB) was studied in an in vitro brain slice preparation. Whole-cell patch recordings were made and spontaneous synaptic responses studied under voltage clamp during application of P2X receptor agonists. ATPγS (100 μ m ) had no effect on holding current, but facilitated spontaneous excitatory postsynaptic current (sEPSC) frequency in 41% of recordings and facilitated spontaneous inhibitory postsynaptic currents (sIPSCs) in 20% of recordings. These were blocked by the P2 receptor antagonist suramin (100 μ m ). α,β-meATP also facilitated sEPSC and sIPSC frequency, while l -β,γ-meATP facilitated only sIPSCs. The sEPSC facilitation by ATPγS was blocked by TTX (but did not block facilitation of sIPSCs). sEPSC facilitation was blocked by PPADS (30 μ m ) and the selective P2X₃ receptor antagonist A-317491 (3 μ m ), suggesting that modulation of sEPSCs involves P2X₃ receptor subunits. α,β-meATP-facilitated sIPSCs were also recorded in wild-type mouse MNTB neurones, but were absent in the MNTB from P2X₁ receptor-deficient mice demonstrating a functional role for P2X₁ receptors in the CNS.     

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.     

Unresolved issues and controversies in purinergic signalling

Some areas of current interest in the rapidly expanding purinergic signalling field that are controversial or are unresolved are highlighted in this review. These include the mechanisms underlying: ATP transport across cell and vesicle membranes; the interaction of multiple receptors for purines and pyrimidines on single cells; the blocking effect of antagonists to P2X₄ and P2X₇ receptors expressed by microglial cells in neuropathic and inflammatory pain; and the complex actions mediated by P2X₇ receptors. Some desirable areas for further research are also discussed including: comparative studies of the evolution of purinergic signalling; studies of purinergic signalling in development and regeneration, including the involvement of stem cells; behavioural studies; and therapeutic strategies.     

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.     

Density-dependent changes of the pore properties of the P2X2 receptor channel

Ligand-gated ion channels underlie and play important roles in synaptic transmission, and it is generally accepted that the ion channel pores have a rigid structure that enables strict regulation of ion permeation. One exception is the P2X ATP-gated channel. After application of ATP, the ion selectivity of the P2X₂ channel time-dependently changes, i.e. permeability to large cations gradually increases, and there is significant cell-to-cell variation in the intensity of inward rectification. Here we show P2X₂ channel properties are correlated with the expression level: increasing P2X₂ expression level in oocytes increases permeability to large cations, decreases inward rectification and increases ligand sensitivity. We also observed that the inward rectification changed in a dose-dependent manner, i.e. when low concentration of ATP was applied to an oocyte with a high expression level, the intensity of inward rectification of the evoked current was weak. Taken together, these results show that the pore properties of P2X₂ channel are not static but change dynamically depending on the open channel density. Furthermore, we identified by mutagenesis study that Ile328 located at the outer mouth of the pore is critical for the density-dependent changes of P2X₂. Our findings suggest synaptic transmission can be modulated by the local density-dependent changes of channel properties caused, for example, by the presence of clustering molecules.     

Dynamic aspects of functional regulation of the ATP receptor channel P2X2

The P2X₂ channel is a ligand-gated channel activated by ATP. Functional features that reflect the dynamic flexibility of the channel include time-dependent pore dilatation following ATP application and direct inhibitory interaction with activated nicotinic acetylcholine receptors on the membrane. We have been studying the mechanisms by which P2X₂ channel functionality is dynamically regulated. Using a Xenopus oocyte expression system, we observed that the pore properties, including ion selectivity and rectification, depend on the open channel density on the membrane. Pore dilatation was apparent when the open channel density was high and inward rectification was modest. We also observed that P2X₂ channels show voltage dependence, despite the absence of a canonical voltage sensor. At a semi-steady state after ATP application, P2X₂ channels were activated upon membrane hyperpolarization. This voltage-dependent activation was also [ATP] dependent. With increases in [ATP], the speed of hyperpolarization-induced activation was increased and the conductance–voltage relationship was shifted towards depolarized potentials. Based on analyses of experimental data and various simulations, we propose that these phenomena can be explained by assuming a fast ATP binding step and a rate-limiting voltage-dependent gating step. Complete elucidation of these regulatory mechanisms awaits dynamic imaging of functioning P2X₂ channels.     

ATP participates in three excitatory postsynaptic potentials in the submucous plexus of the guinea pig ileum

Synaptic transmission between neurones intrinsic to the wall of the intestine involves multiple neurotransmitters. This study aimed to identify neurotransmitters responsible for non-cholinergic excitatory synaptic transmission in the submucous plexus of the guinea pig ileum. Intracellular recordings were made from secretomotor and vasodilator neurones. A single electrical stimulus to a fibre tract evoked excitatory postsynaptic potentials (EPSPs) with three different time courses – fast, slow and an EPSP with an intermediate time course (latency 96 ms, duration 1.2 s). In all neurones, blocking nicotinic receptors reduced fast EPSPs, but they were abolished in only 57 of 78 neurones. Fast EPSPs were also reduced by P2 purinoceptor blockade (5 of 27 neurones) or 5-HT₃ receptor blockade (3 of 20 neurones). The intermediate EPSP was abolished by P2 receptor blockade (13 of 13 neurones) or by the specific P2Y₁ receptor antagonist MRS 2179 (5 of 5 neurones) and was always preceded by a nicotinic or mixed nicotinic/purinergic fast EPSP. Intermediate EPSPs were observed in over half of all neurones including most non-cholinergic secretomotor neurones identified by immunoreactivity for vasoactive intestinal peptide. The slow EPSP evoked by a single pulse stimulus was also abolished by P2 receptor blockade (5 of 5 neurones) or by MRS 2179 (3 of 3 neurones). We conclude that fast EPSPs in submucous neurones are mediated by acetylcholine acting at nicotinic receptors, ATP acting at P2X receptors and 5-HT acting at 5-HT₃ receptors. Both the intermediate EPSP and the single stimulus slow EPSP are mediated by ATP acting at P2Y₁ receptors.     

Localization and function of ATP and GABAA receptors expressed by nociceptors and other postnatal sensory neurons in rat

The role of endogenous GABA and ATP in regulating transmitter release from primary afferent terminals in the superficial dorsal horn of the spinal cord is still controversial. ATP is co-released with GABA from some inhibitory dorsal horn neurons raising the possibility that ATP could act in concert with GABA to regulate transmitter release from primary afferent terminals if receptors to both transmitters are expressed there. Using electrophysiology together with immunocytochemistry, we have investigated the expression of ATP-gated P2X and GABA_A receptors by identified subpopulations of dorsal root ganglion (DRG) neurons known to project primarily to the superficial dorsal horn. Expression of the heat-sensitive vanilloid receptor 1 (VR1) and sensitivity to capsaicin were used to characterize DRG neurons sensitive to noxious heat. Both P2X and GABA_A receptors were expressed on the majority of DRG neurons examined. Recording compound action potentials (CAPs) from dorsal roots in the presence of muscimol, α,β-methylene-ATP (α,β-meATP) or capsaicin resulted in depression of CAP in the slow and medium conducting fibres, indicating cognate receptor expression on the small diameter axons. Dorsal root-evoked dorsal root potentials (DR-DRPs), reflecting depolarization of primary afferent terminals by endogenously released substances, were depressed by the GABA_A receptor antagonist SR95531 and α,β-meATP. These results suggest that GABA_A and P2X receptors are expressed on DRG cell bodies and slow fibre axons, many of which are heat-nociceptive. These fibres project to the superficial lamina of the dorsal horn where the receptors may function to modulate transmitter release near their central terminals.     

Slow excitatory synaptic transmission mediated by P2Y1 receptors in the guinea-pig enteric nervous system

Electrophysiological recording was used to study a type of slow excitatory postsynaptic potential (slow EPSP) that was mediated by release of ATP and its action at P2Y₁ receptors on morphologically identified neurones in the submucosal plexus of guinea-pig small intestine. MRS2179, a selective P2Y₁ purinergic receptor antagonist, blocked both the slow EPSP and mimicry of the EPSP by exogenously applied ATP. Increased conductance accounted for the depolarization phase of the EPSP, which occurred exclusively in neurones with S-type electrophysiological behaviour and uniaxonal morphology. The purinergic excitatory input to the submucosal neurones came from neighbouring neurones in the same plexus, from neurones in the myenteric plexus and from sympathetic postganglionic neurones. ATP-mediated EPSPs occurred coincident with fast nicotinic synaptic potentials evoked by the myenteric projections and with noradrenergic IPSPs evoked by sympathetic fibres that innervated the same neurones. The P2Y₁ receptor on the neurones was identified as a metabotropic receptor linked to activation of phospholipase C, synthesis of inositol 1,4,5-trisphosphate and mobilization of Ca²⁺ from intracellular stores.     

Activity-dependent bidirectional regulation of GABAA receptor channels by the 5-HT4 receptor-mediated signalling in rat prefrontal cortical pyramidal neurons

Emerging evidence has implicated a potential role for 5-HT₄ receptors in cognition and anxiolysis. One of the main target structures of 5-HT₄ receptors on 'cognitive and emotional' pathways is the prefrontal cortex (PFC). As GABAergic signalling plays a key role in regulating PFC functions, we examined the effect of 5-HT₄ receptors on GABA_A receptor channels in PFC pyramidal neurons. Application of 5-HT₄ receptor agonists produced either an enhancement or a reduction of GABA-evoked currents in PFC neurons, which are both mediated by anchored protein kinase A (PKA). Although PKA phosphorylation of GABA_A receptor β3 or β1 subunits leads to current enhancement or reduction respectively in heterologous expression systems, we found that β3 and β1 subunits are co-expressed in PFC pyramidal neurons. Interestingly, altering PKA activation levels can change the direction of the dual effect, switching enhancement to reduction and vice versa. In addition, increased neuronal activity in PFC slices elevated the PKA activation level, changing the enhancing effect of 5-HT₄ receptors on the amplitude of GABAergic inhibitory postsynaptic currents (IPSCs) to a reduction. These results suggest that 5-HT₄ receptors can modulate GABAergic signalling bidirectionally, depending on the basal PKA activation levels that are determined by neuronal activity. This modulation provides a unique and flexible mechanism for 5-HT₄ receptors to dynamically regulate synaptic transmission and neuronal excitability in the PFC network.     

NR2B- and NR2D-containing synaptic NMDA receptors in developing rat substantia nigra pars compacta dopaminergic neurones

NMDA receptors are present at glutamatergic synapses throughout the brain, and are important for the development and plasticity of neural circuits. Their subunit composition is developmentally regulated. We have investigated the developmental profile of functional synaptic NMDA receptor subunits in dopaminergic neurones of the substantia nigra pars compacta (SNc). In SNc dopaminergic neurones from rats aged postnatal day (P)7, ifenprodil inhibited NMDA-EPSCs with an estimated IC₅₀ of 0.36 μ m and a maximum inhibition of 73.5 ± 2.7% (10 μ m ), consistent with a substantial population of NR1/NR2B-containing diheteromeric receptors. UBP141, a novel NR2D-preferring antagonist, inhibited NMDA-EPSCs with an estimated IC₅₀ of 6.2 μ m . During postnatal development, the maximum inhibitory effect of 10 μ m ifenprodil significantly decreased. However, NMDA-EPSCs were not inhibited by Zn²⁺ (200 n m ) or potentiated by the Zn²⁺ chelator TPEN (1 μ m ), and the effect of UBP141 did not increase during development, indicating that NR2B subunits are not replaced with diheteromeric NR2A or NR2D subunits. The time course of the decay of NMDA-EPSCs was not significantly changed in ifenprodil at any age tested. Together, these data suggest that diheteromeric NR1/NR2A or NR1/NR2D receptors do not account for the ifenprodil-resistant component of the NMDA-EPSC. We propose that NR1/NR2B/NR2D triheteromers form a significant fraction of synaptic NMDA receptors during postnatal development. This is the first report of data suggesting NR2D-containing triheteromeric NMDA receptors at a brain synapse.     

Modulation of excitatory synaptic transmission in the spinal substantia gelatinosa of mice deficient in the kainate receptor GluR5 and/or GluR6 subunit

Functional kainate (KA) receptors (KARs) are expressed in the spinal cord substantia gelatinosa (SG) region, and their activation has a capacity to modulate excitatory synaptic transmission at primary afferent synapses with SG neurones. In the present study, we have used gene-targeted mice lacking KAR GluR5 and/or GluR6 subunits to determine the identity of the receptor subunits involved in the KA-induced modulation of excitatory transmission. Our findings reveal that KARs comprising GluR5 or GluR6 subunits can either suppress or facilitate glutamatergic excitatory transmission in the SG of acutely prepared adult mouse spinal cord slices. In the absence of synaptic inhibition mediated by GABA_A and glycine receptors, a biphasic effect of kainate is characteristic with facilitation apparent at a low concentration (30 n m ) and depression at a higher concentration (3 μ m ). In addition, GluR6-KARs, localizing pre- and postsynaptically, are critically involved in inhibiting transmission at both Aδ and C fibre monosynaptic pathways, whereas presynaptic GluR5-KARs play a limited role in inhibiting the C fibre-activated pathway. The results obtained support the hypothesis that KARs are involved in bi-directional regulation of excitatory synaptic transmission in the spinal cord SG region, and that these actions may be of critical importance for nociception and the clinical treatment of pain.     

Serotonin suppresses the slow afterhyperpolarization in rat intralaminar and midline thalamic neurones by activating 5-HT7 receptors

While the highest expression level of 5-HT₇ receptors in the brain is observed in intralaminar and midline thalamic neurones, the physiological role of these receptors in this structure is unknown. In vivo recordings have shown that stimulation of the serotonergic raphe nuclei can alter the response of these neurones to a nociceptive stimulus, suggesting that serotonin modulates their firing properties. Using the patch-clamp technique in rat thalamic brain slices, we demonstrate that activation of 5-HT₇ receptors can strongly modulate the excitability of intralaminar and midline thalamic neurones by inhibiting the calcium-activated potassium conductance that is responsible for the slow afterhyperpolarization (sAHP) following a spike discharge. This sAHP was inhibited after activation of the cAMP pathway, either by bath application of forskolin or intracellular perfusion with 8-bromo-cAMP. The inhibitory effect of 5-HT₇ receptors on sAHPs was blocked by the protein kinase A antagonist R _P -cAMPS. Calcium-imaging experiments showed no change in intracellular calcium levels during the 5-HT₇ response, indicating that in these neurones, a global calcium signal was not necessary to activate the cAMP cascade. Finally, bath application of serotonin produced a strong increase in cytosolic cAMP concentration, as measured using the fluorescent probe FlCRhR, and an inhibition of the sAHP. Taken together, these results suggest that 5-HT₇ receptors are implicated in the effect of 5-HT on sAHP in intralaminar and midline thalamic neurones, an effect that is mediated by the cAMP second-messenger cascade.     

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.     

Hypo-osmotic potentiation of acetylcholine-stimulated ciliary beat frequency through ATP release in rat tracheal ciliary cells

The ciliary beat frequency (CBF) of rat tracheal ciliary cells in a slice preparation was measured using video-enhanced contrast (VEC) microscopy. Acetylcholine (ACh) increased CBF mediated via intracellular Ca²⁺ concentration ([Ca²⁺]_i) in a dose-dependent manner. An adequate hypo-osmotic stress (−40 mos m ) potentiated ACh-stimulated CBF increase in tracheal ciliary cells and shifted the ACh dose–response curve to the left (lower concentration side). This potentiation was independent of hypo-osmotic stresses applied ranging from −20 mosM to −90 mosM. A hypo-osmotic stress induces ATP release in many cell types. The present study demonstrated that suramin (an inhibitor of purinergic receptors) and apyrase (an ATPase/ADPase) eliminate the hypo-osmotic potentiation of ACh-stimulated CBF increase and that ATP increased [Ca²⁺]_i and CBF, as well as potentiating ACh-stimulated rises in [Ca²⁺]_i and CBF increase. Moreover, the apical surface of tracheal ciliary cells were stained immunopositive for the P2X₄ purinergic receptor. A hypo-osmotic stress (−40 mosM) transiently increased [Ca²⁺]_i and potentiated the ACh-stimulated [Ca²⁺]_i increase. The hypo-osmotic potentiation of ACh-stimulated CBF increase was not detected under Ca²⁺-free conditions. These observations suggest that a hypo-osmotic stress stimulates ATP release from the trachea. The released ATP may induce further increases in [Ca²⁺]_i and CBF in ACh-stimulated tracheal ciliary cells, which may be mediated by purinergic receptors, such as P2X₄.     

Binding and direct activation of the epithelial Na+ channel (ENaC) by phosphatidylinositides

Several distinct types of ion channels bind and directly respond to phosphatidylinositides, including phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P₃) and phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P₂). This regulation is physiologically relevant for its dysfunction, in some instances, causes disease. Recent studies identify the epithelial Na⁺ channel (ENaC) as a channel sensitive to phosphatidylinositides. ENaC appears capable of binding both PI(4,5)P₂ and PI(3,4,5)P₃ with binding stabilizing channel gating. The binding sites for these molecules within ENaC are likely to be distinct with the former phosphoinositide interacting with elements in the cytosolic NH₂-terminus of the β- and γ-ENaC subunits and the latter with cytosolic regions immediately following the second transmembrane domains in these two subunits. PI(4,5)P₂ binding to ENaC appears saturated at rest and necessary for channel gating. Thus, decreases in cellular PI(4,5)P₂ levels may serve as a convergence point for inhibitory regulation of ENaC by G-protein coupled receptors and receptor tyrosine kinases. In contrast, apparent PI(3,4,5)P₃ binding to ENaC is not saturated. This enables the channel to respond with gating changes in a rapid and dynamic manner to signalling input that influences cellular PI(3,4,5)P₃ levels.     

Cannabinoid-induced presynaptic inhibition at the primary afferent trigeminal synapse of juvenile rat brainstem slices

Systemic or intraventricular administration of cannabinoids causes analgesic effects, but relatively little is known for their cellular mechanism. Using brainstem slices with the mandibular nerve attached, we examined the effect of cannabinoids on glutamatergic transmission in superficial trigeminal caudal nucleus of juvenile rats. The exogenous cannabinoid receptor agonist WIN 55,212-2 (WIN), as well as the endogenous agonist anandamide, hyperpolarized trigeminal caudal neurones and depressed the amplitude of excitatory postsynaptic potentials (EPSPs) or currents (EPSCs) monosynaptically evoked by stimulating mandibular nerves in a concentration-dependent manner. The inhibitory action of WIN was blocked or fully reversed by the CB₁ receptor antagonist SR 141716A. WIN had no effect on the amplitude of miniature excitatory postsynaptic currents (mEPSCs) recorded in the presence of tetrodotoxin or cadmium. The inhibitory effect of WIN on EPSCs was greater for those with longer synaptic latency, suggesting that cannabinoids have a stronger effect on C-fibre EPSPs than on Aδ-fibre EPSPs. Ba²⁺ (100 μ m ) blocked the hyperpolarizing effect of cannabinoids, but did not affect their inhibitory effect on EPSPs. The N-type Ca²⁺ channel blocker ω-conotoxin GVIA (ω-CgTX) occluded the WIN-mediated presynaptic inhibition, whereas the P/Q-type Ca²⁺ channel blocker ω-agatoxin TK (ω-Aga) had no effect. These results suggest that cannabinoids preferentially activate CB₁ receptors at the nerve terminal of small-diameter primary afferent fibres. Upon activation, CB₁ receptors may selectively inhibit presynaptic N-type Ca²⁺ channels and exocytotic release machinery, thereby attenuating the transmitter release at the trigeminal nociceptive synapses.     

Intracellular calcium signalling in magnocellular neurones of the rat supraoptic nucleus: understanding the autoregulatory mechanisms

Oxytocin and vasopressin, released at the soma and dendrites of neurones, bind to specific autoreceptors and induce an increase in [Ca²⁺]₁. In oxytocin cells, the increase results from a mobilisation of Ca²⁺ from intracellular stores, whereas in vasopressin cells, it results mainly from an influx of Ca²⁺ through voltage-dependent channels. The response to vasopressin is coupled to phospholipase C and adenylyl-cyclase pathways which are activated by V₁ (V_1a and V_1b)- and V₂-type receptors respectively. Measurements of [Ca²⁺]₁ in response to V_1a and V₂ agonists and antagonists suggest the functional expression of these two types of receptors in vasopressin neurones. The intracellular mechanisms involved are similar to those observed for the action of the pituitary adenylyl-cyclase-activating peptide (PACAP). Isolated vasopressin neurones exhibit spontaneous [Ca²⁺]₁ oscillations and these are synchronised with phasic bursts of electrical activity. Vasopressin modulates these spontaneous [Ca²⁺]₁ oscillations in a manner that depends on the initial state of the neurone, and such varied effects of vasopressin may be related to those observed on the electrical activity of vasopressin neurones in vivo.