Extracellular ATP inhibits excitatory synaptic input on parvalbumin positive interneurons and attenuates gamma oscillations via P2X4 receptors.

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

Title: P2x7 Receptor Activation and Estrogen Status Drive Neuroinflammatory Mechanisms in a Rat Model for Dry Eye.

Dry eye disease (DED) is recognized as a chronic inflammatory condition with an increase in tear osmolarity and loss of tear film integrity. DED is often accompanied by adverse ocular symptoms which are more prevalent in females than males. The basis for ocular hyperalgesia in DED remains uncertain; however, both peripheral and central neural mechanisms are implicated. A model for aqueous deficient DED, exorbital gland excision, was used to determine if activation of the purinergic receptor subtype 7, P2X7R, expressed by non-neural cells in peripheral and central trigeminal nerve pathways, contributed to persistent ocular hyperalgesia. Densitometry of trigeminal brainstem sections revealed increases in P2X7R, the myeloid cell marker Iba1, and the inflammasome, NLRP3, of estradiol-treated DED females compared to estradiol-treated sham females, while expression in DED males and DED females not given estradiol displayed minor changes. No evidence of immune cell infiltration into the trigeminal brainstem was seen in DED rats; however, markers for microglia activation (Iba1) were increased in all groups. Isolated microglia expressed increased levels of P2X7R and P2X4R, IL-1ß (Ιnterleukin-1ß), NLRP3, and iNOS (nitric oxide synthase). Further, estradiol-treated DED females displayed greater increases in P2X7R, IL-1ß and NLRP3 expression compared to untreated DED females. Orbicularis oculi muscle activity (OOemg) evoked by ocular instillation of hypertonic saline (HS) was recorded as a surrogate measure of ocular hyperalgesia and was markedly enhanced in all DED groups compared to sham rats. Systemic minocycline reduced HS-evoked OOemg in all DED groups compared to sham rats. Local microinjection in the caudal trigeminal brainstem of an antagonist for P2X7R (A804598) greatly reduced HS-evoked OOemg activity in all DE groups, while responses in sham groups were not affected. Intra-trigeminal ganglion injection of siRNA for P2X7R significantly reduced HS-evoked OOemg activity in all DED groups, while evoked responses in sham animals were not affected. These results indicated that activation of P2X7R at central and peripheral sites in trigeminal pain pathways contributed to an increase in ocular hyperalgesia and microglia activation in DED males and females. Estrogen treatment in females further amplified ocular hyperalgesia and neuroimmune responses in this model for aqueous deficient DED.

Lithocholic acid inhibits P2X2 and potentiates P2X4 receptor channel gating.

The family of ATP-gated purinergic P2X receptors comprises seven bunits (P2X1-7) that are unevenly distributed in the central and peripheral nervous systems as well as other organs. Endogenous modulators of P2X receptors are phospholipids, steroids and neurosteroids. Here, we analyzed whether bile acids, which are natural products derived from cholesterol, affect P2X receptor activity. We examined the effects of primary and secondary bile acids and newly synthesized derivatives of lithocholic acid on agonist-induced responses in HEK293T cells expressing rat P2X2, P2X4 and P2X7 receptors. Electrophysiology revealed that low micromolar concentrations of lithocholic acid and its structural analog 4-dafachronic acid strongly inhibit ATP-stimulated P2X2 but potentiate P2X4 responses, whereas primary bile acids and other secondary bile acids exhibit no or reduced effects only at higher concentrations. Agonist-stimulated P2X7 responses are significantly potentiated by lithocholic acid at moderate concentrations. Structural modifications of lithocholic acid at positions C-3, C-5 or C-17 abolish both inhibitory and potentiation effects to varying degrees, and the 3α-hydroxy group contributes to the ability of the molecule to switch between potentiation and inhibition. Lithocholic acid allosterically modulates P2X2 and P2X4 receptor sensitivity to ATP, reduces the rate of P2X4 receptor desensitization and antagonizes the effect of ivermectin on P2X4 receptor deactivation. Alanine-scanning mutagenesis of the upper halve of P2X4 transmembrane domain-1 revealed that residues Phe48, Val43 and Tyr42 are important for potentiating effect of lithocholic acid, indicating that modulatory sites for lithocholic acid and ivermectin partly overlap. Lithocholic acid also inhibits ATP-evoked currents in pituitary gonadotrophs expressing native P2X2, and potentiates ATP currents in nonidentified pituitary cells expressing P2X4 receptors. These results indicate that lithocholic acid is a bioactive steroid that may help to further unveil the importance of the P2X2, and P2X4 receptors in many physiological processes.

Full-Length P2X7 Structures Reveal How Palmitoylation Prevents Channel Desensitization.

P2X receptors are trimeric, non-selective cation channels activated by extracellular ATP. The P2X7 receptor subtype is a pharmacological target because of involvement in apoptotic, inflammatory, and tumor progression pathways. It is the most structurally and functionally distinct P2X subtype, containing a unique cytoplasmic domain critical for the receptor to initiate apoptosis and not undergo desensitization. However, lack of structural information about the cytoplasmic domain has hindered understanding of the molecular mechanisms underlying these processes. We report cryoelectron microscopy structures of full-length rat P2X7 receptor in apo and ATP-bound states. These structures reveal how one cytoplasmic element, the C-cys anchor, prevents desensitization by anchoring the pore-lining helix to the membrane with palmitoyl groups. They show a second cytoplasmic element with a unique fold, the cytoplasmic ballast, which unexpectedly contains a zinc ion complex and a guanosine nucleotide binding site. Our structures provide first insights into the architecture and function of a P2X receptor cytoplasmic domain.

Integration of purinergic and angiotensin II receptor function in renal vascular responses and renal injury in angiotensin II-dependent hypertension.

Glomerular arteriolar vasoconstriction and tubulointerstitial injury are observed before glomerular damage occurs in models of hypertension. High interstitial ATP concentrations, caused by the increase in arterial pressure, alter renal mechanisms involved in the long-term control of blood pressure, autoregulation of glomerular filtration rate and blood flow, tubuloglomerular feedback (TGF) responses, and sodium excretion. Elevated ATP concentrations and augmented expression of P2X receptors have been demonstrated under a genetic background or induction of hypertension with vasoconstrictor peptides. In addition to the alterations of the microcirculation in the hypertensive kidney, the vascular actions of elevated intrarenal angiotensin II levels may be mitigated by the administration of broad purinergic P2 antagonists or specific P2Y12, P2X1, and P2X7 receptor antagonists. Furthermore, the prevention of tubulointerstitial infiltration with immunosuppressor compounds reduces the development of salt-sensitive hypertension, indicating that tubulointerstitial inflammation is essential for the development and maintenance of hypertension. Inflammatory cells also express abundant purinergic receptors, and their activation by ATP induces cytokine and growth factor release that in turn contributes to augment tubulointerstitial inflammation. Collectively, the evidence suggests a pathophysiological activation of purinergic P2 receptors in angiotensin-dependent hypertension. Coexistent increases in intrarenal angiotensin II and activates Ang II AT1 receptors, which interacts with over-activated purinergic receptors in a complex manner, suggesting convergence of their post-receptor signaling processes.

Neonatal Maternal Deprivation Enhances Presynaptic P2X7 Receptor Transmission in Insular Cortex in an Adult Rat Model of Visceral Hypersensitivity.

AIMS: Insular cortex (IC) is involved in processing the information of pain. The aim of this study was to investigate roles and mechanisms of P2X7 receptors (P2X7Rs) in IC in development of visceral hypersensitivity of adult rats with neonatal maternal deprivation (NMD). METHODS: Visceral hypersensitivity was quantified by abdominal withdrawal reflex threshold to colorectal distension (CRD). Expression of P2X7Rs was determined by qPCR and Western blot. Synaptic transmission in IC was recorded by patch-clamp recording. RESULTS: The expression of P2X7Rs and glutamatergic neurotransmission in IC was significantly increased in NMD rats when compared with age-matched controls. Application of BzATP (P2X7R agonist) enhanced the frequency of spontaneous excitatory postsynaptic currents (sEPSC) and miniature excitatory postsynaptic currents (mEPSC) in IC slices of control rats. Application of BBG (P2X7R antagonist) suppressed the frequencies of sEPSC and mEPSC in IC slices of NMD rats. Microinjection of BzATP into right IC significantly decreased CRD threshold in control rats while microinjection of BBG or A438079 into right IC greatly increased CRD threshold in NMD rats. CONCLUSION: Data suggested that the enhanced activities of P2X7Rs in IC, likely through a presynaptic mechanism, contributed to visceral hypersensitivity of adult rats with NMD.

Hydrogen sulfide inhibits adenosine triphosphate-induced activation and IL-1βreleases in rat microglia / 中国病理生理杂志

AIM: To investigate the effects of sodium hydrosulfide ( NaHS ) , a donor of hydrogen sulfide ( H2 S) , on the membrane permeability , intracellular Ca 2+concentration ( [ Ca2+] i ) and the release of IL-1βinduced by a-denosine triphosphate (ATP) in rat microglia, and to explore the effect of H2S on ATP-P2X purinergic signaling pathway and the molecular mechanism of its neuroprotective effect .METHODS: Rat microglia in logarithmic growth phase were used in the study.The [Ca2+]i was detected by Fura-2/AM staining.Fluorescent dye YO-PRO-1 was used to observe the membrane permeability.Interleukin-1β(IL-1β) was measured by rat IL-1βELISA kits.RESULTS:The YO-PRO-1 flu-orescence intensity was obviously elevated by ATP induction in a dose -dependent manner in the rat microglia , but this effect was counteracted by NaHS pretreatment (P<0.05).[Ca2+]i rapidly increased and then decreased slowly , forming a sta-ble platform for a long time when rat microglia were treated with ATP .Ca2+spike activity induced by ATP had no change , but the platform disappeared (P<0.05) after NaHS pretreatment.The ATP and LPS together facilitated the release of IL-1β, but the phenomenon was inhibited by NaHS (P<0.05).CONCLUSION:Hydrogen sulfide may decrease the mem-brane permeability , calcium inflow and IL-1βrelease in rat microglia activated by high dose of ATP .The cytoprotection of hydrogen sulfide may be mediated by purinergic signaling pathway .

The effect of 5-HT and electrical field stimulation on the contractility of the whole isolated urinary bladder of Suncus murinus.

The present study used the whole isolated urinary bladder of Suncus murinus, to investigate the effect of exogenously added serotonin (5-HT) and electrical field stimulation (EFS) in the absence and presence of methysergide, a 5-HT1/2/7 receptor antagonist or the selective 5-HT7 receptor antagonist, SB269970. Further experiments investigated the involvement of potassium channel, cholinergic and purinergic systems in mediating the contractile response to EFS. Pre-treatment with methysergide reduced and increased the contractile responses to 5-HT and EFS, respectively. Pre-treatment with SB269970 increased the responses to 5-HT without modifying the EFS-induced contractions. EFS-induced contractions were not modified by pre-treatment with atropine (10µM), α-ß-methylene ATP or glibenclamide. EFS-induced contractions were attenuated by cromakalim (10µM) or atropine (0.1 µM). In conclusion, the 5-HT2 receptors are likely to play a role in mediating the contractile response to 5-HT in detrusor muscle. Furthermore, EFS-induced contractions are mediated through cholinergic and an unknown neurotransmitter which is modulated by K(ATP) channels in the detrusor muscle of Suncus murinus.