Neurosteroids are steroids synthesized de novo in the brain from cholesterol in an independent manner from peripheral steroid sources. The term "neuroactive steroid" includes all steroids independent of their origin, and newly synthesized analogs of neurosteroids that modify neuronal activities. In vivo application of neuroactive steroids induces potent anxiolytic, antidepressant, anticonvulsant, sedative, analgesic and amnesic effects, mainly through interaction with the γ-aminobutyric acid type-A receptor (GABAAR). However, neuroactive steroids also act as positive or negative allosteric regulators on several ligand-gated channels including N-methyl-d-aspartate receptors (NMDARs), nicotinic acetylcholine receptors (nAChRs) and ATP-gated purinergic P2X receptors. Seven different P2X subunits (P2X1-7) can assemble to form homotrimeric or heterotrimeric ion channels permeable for monovalent cations and calcium. Among them, P2X2, P2X4, and P2X7 are the most abundant within the brain and can be regulated by neurosteroids. Transmembrane domains are necessary for neurosteroid binding, however, no generic motif of amino acids can accurately predict the neurosteroid binding site for any of the ligand-gated ion channels including P2X. Here, we will review what is currently known about the modulation of rat and human P2X by neuroactive steroids and the possible structural determinants underlying neurosteroid-induced potentiation and inhibition of the P2X2 and P2X4 receptors. This article is part of the Special Issue on "Purinergic Signaling: 50 years".

• MeSH
• adenosintrifosfát metabolismus   MeSH
• iontové kanály řízené ligandy * metabolismus   MeSH
• krysa rodu rattus MeSH
• lidé MeSH
• mozek metabolismus   MeSH
• neurosteroidy * MeSH
• purinergní receptory P2X metabolismus   MeSH
• purinergní receptory P2X2 metabolismus   MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

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.

• MeSH
• adenohypofýza cytologie   účinky léků   fyziologie   MeSH
• gating iontového kanálu účinky léků   MeSH
• HEK293 buňky MeSH
• hypothalamus cytologie   účinky léků   fyziologie   MeSH
• kyselina lithocholová analogy a deriváty   farmakologie   MeSH
• lidé MeSH
• neurony účinky léků   fyziologie   MeSH
• potkani Wistar MeSH
• purinergní receptory P2X - agonisté farmakologie   MeSH
• purinergní receptory P2X - antagonisté farmakologie   MeSH
• purinergní receptory P2X2 fyziologie   MeSH
• purinergní receptory P2X4 fyziologie   MeSH
• purinergní receptory P2X7 fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

P2X receptors (P2XRs) are ATP-gated cationic channels that are allosterically modulated by numerous compounds, including steroids and neurosteroids. These compounds may both inhibit and potentiate the activity of P2XRs, but sex steroids such as 17β-estradiol or progesterone are reported to be inactive. Here, we tested a hypothesis that testosterone, another sex hormone, modulates activity of P2XRs. We examined actions of native testosterone and a series of testosterone derivatives on the gating of recombinant P2X2R, P2X4R and P2X7R and native channels expressed in pituitary cells and hypothalamic neurons. The 17β-ester derivatives of testosterone rapidly and positively modulate the 1 µM ATP-evoked currents in P2X2R- and P2X4R-expressing cells, but not agonist-evoked currents in P2X7R-expressing cells. In general, most of the tested testosterone derivatives are more potent modulators than endogenous testosterone. The comparison of chemical structures and whole-cell recordings revealed that their interactions with P2XRs depend on the lipophilicity and length of the alkyl chain at position C-17. Pre-treatment with testosterone butyrate or valerate increases the sensitivity of P2X2R and P2X4R to ATP by several fold, reduces the rate of P2X4R desensitization, accelerates resensitization, and enhances ethidium uptake by P2X4R. Native channels are also potentiated by testosterone derivatives, while endogenously expressed GABA receptors type A are inhibited. The effect of ivermectin, a P2X4R-specific allosteric modulator, on deactivation is antagonized by testosterone derivatives in a concentration-dependent manner. Together, our results provide evidence for potentiation of particular subtypes of P2XRs by testosterone derivatives and suggest a potential role of ivermectin binding site for steroid-induced modulation. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

• MeSH
• gating iontového kanálu účinky léků   fyziologie   MeSH
• HEK293 buňky MeSH
• krysa rodu rattus MeSH
• lidé MeSH
• potkani Wistar MeSH
• purinergní receptory P2X2 metabolismus   MeSH
• purinergní receptory P2X4 metabolismus   MeSH
• testosteron farmakologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The supraoptic nuclei (SON), the hypothalamic release site of vasopressin and oxytocin, receive a non-glutamatergic, excitatory input from the caudal medulla that uses noradrenaline and ATP as neurotransmitters. Here, we studied the actions of extracellular ATP on SON neurons in hypothalamic slices isolated from the brains of 16- to 24-day-old rats. Whole-cell current clamp recordings performed 1-6 h after isolation showed that exogenous ATP application increased the frequency of action potentials and induced the depolarization of resting membranes. Voltage clamp recordings showed that ATP increased the frequency of GABAergic or glutamatergic spontaneous synaptic currents without changing their amplitude and evoked inward current (126±13 pA) in about 80% of SON neurons. The application of ATPγS and 2MeSATP mimicked the effects of ATP, but 2MeSADP, 2MeSAMP and αβmeATP had no effect. The P2X7 receptor agonist, BzATP, did not induce an inward current, but it increased intracellular calcium concentration in non-neuronal SON cells in slices. Suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) inhibited ATP-induced currents, whereas pH 6.5 and ivermectin, a specific allosteric modulator of the P2X4 receptor, potentiated ATP-induced currents. The P2Y1-selective antagonist, 2'-deoxy-N⁶-methyladenosine 3',5'-bisphosphate tetrasodium salt (MRS 2179), had no effect on ATP-induced responses. Quantitative real-time PCR showed that P2X2>P2X7>P2X4 purinergic receptor mRNAs were expressed in the SON tissue, but the levels of P2X1, P2X3, P2X5, P2X6, P2Y1, P2Y2 and P2Y12 mRNA were minor. These results show that SON neurons express functional presynaptic and extrasynaptic P2X2 and P2X4 receptors that modulate glutamate and GABA release and control the electrical excitability of SON neurons.

• MeSH
• adenosintrifosfát farmakologie   MeSH
• akční potenciály účinky léků   fyziologie   MeSH
• GABA sekrece   MeSH
• krysa rodu rattus MeSH
• kyselina glutamová sekrece   MeSH
• metoda terčíkového zámku MeSH
• neurony účinky léků   sekrece   MeSH
• nucleus supraopticus účinky léků   metabolismus   MeSH
• orgánové kultury - kultivační techniky MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• potkani Wistar MeSH
• purinergní receptory P2X2 metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH