Purinergic P2X receptors (P2X) are ATP-gated ion channels that are broadly expressed in the brain, particularly in the hypothalamus. As ionic channels with high permeability to calcium, P2X play an important and active role in neural functions. The hypothalamus contains a number of small nuclei with many molecularly defined types of peptidergic neurons that affect a wide range of physiological functions, including water balance, blood pressure, metabolism, food intake, circadian rhythm, childbirth and breastfeeding, growth, stress, body temperature, and multiple behaviors. P2X are expressed in hypothalamic neurons, astrocytes, tanycytes, and microvessels. This review focuses on cell-type specific expression of P2X in the most important hypothalamic nuclei, such as the supraoptic nucleus (SON), paraventricular nucleus (PVN), suprachiasmatic nucleus (SCN), anteroventral periventricular nucleus (AVPV), anterior hypothalamic nucleus (AHN), arcuate nucleus (ARC), ventromedial hypothalamic nucleus (VMH), dorsomedial hypothalamic nucleus (DMH), tuberomammillary nucleus (TMN), and lateral hypothalamic area (LHA).> The review also notes the possible role of P2X and extracellular ATP in specific hypothalamic functions. The literature summarized here shows that purinergic signaling is involved in the control of the hypothalamic-pituitary endocrine system, the hypothalamic-neurohypophysial system, the circadian systems and nonendocrine hypothalamic functions.

• Klíčová slova
• P2X, arcuate nucleus (ARC), extracellular ATP, hypothalamus, paraventricular nucleus (PVN), suprachiasmatic nucleus (SCN), supraoptic nucleus (SON),
• MeSH
• adenosintrifosfát metabolismus   MeSH
• astrocyty metabolismus   MeSH
• hypothalamus * metabolismus   cytologie   MeSH
• lidé MeSH
• neurony metabolismus   MeSH
• purinergní receptory P2X * metabolismus   genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• adenosintrifosfát MeSH
• purinergní receptory P2X * MeSH

Activation of the P2X7 receptor results in the opening of a large pore that plays a role in immune responses, apoptosis, and many other physiological and pathological processes. Here, we investigated the role of conserved and unique residues in the extracellular vestibule connecting the agonist-binding domain with the transmembrane domain of rat P2X7 receptor. We found that all residues that are conserved among the P2X receptor subtypes respond to alanine mutagenesis with an inhibition (Y51, Q52, and G323) or a significant decrease (K49, G326, K327, and F328) of 2',3'-O-(benzoyl-4-benzoyl)-ATP (BzATP)-induced current and permeability to ethidium bromide, while the nonconserved residue (F322), which is also present in P2X4 receptor, responds with a 10-fold higher sensitivity to BzATP, much slower deactivation kinetics, and a higher propensity to form the large dye-permeable pore. We examined the membrane expression of conserved mutants and found that Y51, Q52, G323, and F328 play a role in the trafficking of the receptor to the plasma membrane, while K49 controls receptor responsiveness to agonists. Finally, we studied the importance of the physicochemical properties of these residues and observed that the K49R, F322Y, F322W, and F322L mutants significantly reversed the receptor function, indicating that positively charged and large hydrophobic residues are important at positions 49 and 322, respectively. These results show that clusters of conserved residues above the transmembrane domain 1 (K49-Y51-Q52) and transmembrane domain 2 (G326-K327-F328) are important for receptor structure, membrane expression, and channel gating and that the nonconserved residue (F322) at the top of the extracellular vestibule is involved in hydrophobic inter-subunit interaction which stabilizes the closed state of the P2X7 receptor channel.

• Klíčová slova
• HEK293T cells, P2X7 receptor, deactivation, dye uptake, extracellular vestibule, gating, mutagenesis,
• MeSH
• bakteriální proteiny chemie   genetika   metabolismus   MeSH
• gating iontového kanálu MeSH
• HEK293 buňky MeSH
• interakční proteinové domény a motivy MeSH
• kinetika MeSH
• konzervovaná sekvence MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• luminescentní proteiny chemie   genetika   metabolismus   MeSH
• molekulární modely MeSH
• mutageneze cílená MeSH
• mutantní proteiny chemie   genetika   metabolismus   MeSH
• proteinové domény MeSH
• purinergní receptory P2X7 chemie   genetika   metabolismus   MeSH
• rekombinantní fúzní proteiny chemie   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• statická elektřina MeSH
• substituce aminokyselin MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny MeSH
• luminescentní proteiny MeSH
• mutantní proteiny MeSH
• P2rx7 protein, rat MeSH Prohlížeč
• purinergní receptory P2X7 MeSH
• rekombinantní fúzní proteiny MeSH
• yellow fluorescent protein, Bacteria MeSH Prohlížeč

Magnocellular neurons in the supraoptic nucleus (SON), which synthesize and release arginine vasopressin (AVP) and oxytocin (OT), express several subtypes of ATP-stimulated purinergic P2X receptors (P2XR) that modulate neuronal activity as well as neurotransmitter and hormone release. However, the physiological impact of this modulation is not well understood. Here, we tested a hypothesis that P2XRs play a role in the sustained release of hormones from SON neurons stimulated through fasting/refeeding. We studied the effect of 2 h of refeeding after 48 h of fasting on P2XR and P2YR mRNA expression and ATP-induced presynaptic and postsynaptic responses in the SON of 30-day-old rats. Quantitative real-time PCR revealed that the expression of P2X2R and AVP mRNA was upregulated, whereas P2X4R, P2X7R, P2Y2R, and OT mRNA levels were not significantly changed and P2Y1R mRNA expression was decreased. Whole-cell patch clamp recordings performed on isolated rat brain slices showed that the amplitude of the ATP-stimulated somatic current and the ATP-induced increases in the frequency of spontaneous GABAergic inhibitory postsynaptic currents were significantly higher in SON neurons from fasted/refed rats than in SON neurons from normally fed rats. No evidence was found for changes in the presynaptic effect of ATP in SON neurons not expressing somatic P2XRs. These results suggest that the increased activity of SON neurons synthesizing AVP is associated with enhanced expression of P2X2Rs on neuronal cell bodies and their GABAergic presynaptic nerve terminals.

• Klíčová slova
• ATP, GABA, P2X receptor, P2Y receptor, arginine vasopressin, oxytocin, supraoptic nucleus,
• Publikační typ
• časopisecké články MeSH

The circadian rhythms in physiological and behavioral functions are driven by a pacemaker located in the suprachiasmatic nucleus (SCN). The rhythms continue in constant darkness and depend on cell-cell communication between neurons and glia. The SCN astrocytes generate also a circadian rhythm in extracellular adenosine 5'-triphosphate (ATP) accumulation, but molecular mechanisms that regulate ATP release are poorly understood. Here, we tested the hypothesis that ATP is released via the plasma membrane purinergic P2X7 receptors (P2X7Rs) and P2Y receptors (P2YRs) which have been previously shown to be expressed in the SCN tissue at transcriptional level. We have investigated this hypothesis using SCN organotypic cultures, primary cultures of SCN astrocytes, ATP bioluminescent assays, immunohistochemistry, patch-clamping, and calcium imaging. We found that extracellular ATP accumulation in organotypic cultures followed a circadian rhythm, with a peak between 24:00 and 04:00 h, and the trough at ~12:00 h. ATP rhythm was inhibited by application of AZ10606120, A438079, and BBG, specific blockers of P2X7R, and potentiated by GW791343, a positive allosteric modulator of this receptor. Double-immunohistochemical staining revealed high expression of the P2X7R protein in astrocytes of SCN slices. PPADS, a non-specific P2 antagonist, and MRS2179, specific P2Y1R antagonist, also abolished ATP rhythm, whereas the specific P2X4R blocker 5-BDBD was not effective. The pannexin-1 hemichannel blocker carbenoxolone displayed a partial inhibitory effect. The P2Y1R agonist MRS2365, and the P2Y2R agonist MRS2768 potentiated ATP release in organotypic cultures and increase intracellular Ca2+ level in cultured astrocytes. Thus, SCN utilizes multiple purinergic receptor systems and pannexin-1 hemichannels to release ATP.

• Klíčová slova
• ATP release, P2X7 receptor, P2Y1 receptor, P2Y2 receptor, astrocytes, organotypic cultures, pannexin-1 hemichannel, suprachiasmatic nucleus,
• Publikační typ
• časopisecké články MeSH

The hypothalamic suprachiasmatic nuclei (SCN), the circadian master clock in mammals, releases ATP in a rhythm, but the role of extracellular ATP in the SCN is still unknown. In this study, we examined the expression and function of ATP-gated P2X receptors (P2XRs) in the SCN neurons of slices isolated from the brain of 16- to 20-day-old rats. Quantitative RT-PCR showed that the SCN contains mRNA for P2X 1-7 receptors and several G-protein-coupled P2Y receptors. Among the P2XR subunits, the P2X2 > P2X7 > P2X4 mRNAs were the most abundant. Whole-cell patch-clamp recordings from SCN neurons revealed that extracellular ATP application increased the frequency of spontaneous GABAergic IPSCs without changes in their amplitudes. The effect of ATP appears to be mediated by presynaptic P2X2Rs because ATPγS and 2MeS-ATP mimics, while the P2XR antagonist PPADS blocks, the observed enhancement of the frequency of GABA currents. There were significant differences between two SCN regions in that the effect of ATP was higher in the ventrolateral subdivision, which is densely innervated from outside the SCN. Little evidence was found for the presence of P2XR channels in somata of SCN neurons as P2X2R immunoreactivity colocalized with synapsin and ATP-induced current was observed in only 7% of cells. In fura-2 AM-loaded slices, BzATP as well as ADP stimulated intracellular Ca(2+) increase, indicating that the SCN cells express functional P2X7 and P2Y receptors. Our data suggest that ATP activates presynaptic P2X2Rs to regulate inhibitory synaptic transmission within the SCN and that this effect varies between regions.

• MeSH
• adenosintrifosfát farmakologie   MeSH
• antagonisté excitačních aminokyselin farmakologie   MeSH
• biofyzikální jevy účinky léků   MeSH
• blokátory sodíkových kanálů farmakologie   MeSH
• GABA farmakologie   MeSH
• inhibitory agregace trombocytů farmakologie   MeSH
• krysa rodu Rattus MeSH
• kultivované buňky MeSH
• messenger RNA metabolismus   MeSH
• metoda terčíkového zámku MeSH
• nervový přenos účinky léků   MeSH
• nervový útlum účinky léků   MeSH
• neurony účinky léků   MeSH
• novorozená zvířata MeSH
• nucleus suprachiasmaticus cytologie   MeSH
• potkani Wistar MeSH
• purinergní látky farmakologie   MeSH
• purinergní receptory P2X genetika   metabolismus   MeSH
• regulace genové exprese účinky léků   MeSH
• synaptické potenciály účinky léků   MeSH
• techniky in vitro MeSH
• tetrodotoxin farmakologie   MeSH
• vápník metabolismus   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfát MeSH
• antagonisté excitačních aminokyselin MeSH
• blokátory sodíkových kanálů MeSH
• GABA MeSH
• inhibitory agregace trombocytů MeSH
• messenger RNA MeSH
• purinergní látky MeSH
• purinergní receptory P2X MeSH
• tetrodotoxin MeSH
• vápník MeSH

The binding of ATP to trimeric P2X receptors (P2XR) causes an enlargement of the receptor extracellular vestibule, leading to opening of the cation-selective transmembrane pore, but specific roles of vestibule amino acid residues in receptor activation have not been evaluated systematically. In this study, alanine or cysteine scanning mutagenesis of V47-V61 and F324-N338 sequences of rat P2X4R revealed that V49, Y54, Q55, F324, and G325 mutants were poorly responsive to ATP and trafficking was only affected by the V49 mutation. The Y54F and Y54W mutations, but not the Y54L mutation, rescued receptor function, suggesting that an aromatic residue is important at this position. Furthermore, the Y54A and Y54C receptor function was partially rescued by ivermectin, a positive allosteric modulator of P2X4R, suggesting a rightward shift in the potency of ATP to activate P2X4R. The Q55T, Q55N, Q55E, and Q55K mutations resulted in non-responsive receptors and only the Q55E mutant was ivermectin-sensitive. The F324L, F324Y, and F324W mutations also rescued receptor function partially or completely, ivermectin action on channel gating was preserved in all mutants, and changes in ATP responsiveness correlated with the hydrophobicity and side chain volume of the substituent. The G325P mutant had a normal response to ATP, suggesting that G325 is a flexible hinge. A topological analysis revealed that the G325 and F324 residues disrupt a β-sheet upon ATP binding. These results indicate multiple roles of the extracellular vestibule amino acid residues in the P2X4R function: the V49 residue is important for receptor trafficking to plasma membrane, the Y54 and Q55 residues play a critical role in channel gating and the F324 and G325 residues are critical for vestibule widening.

• MeSH
• adenosintrifosfát chemie   metabolismus   MeSH
• alosterická regulace MeSH
• aminokyseliny chemie   genetika   metabolismus   MeSH
• bodová mutace MeSH
• gating iontového kanálu účinky léků   fyziologie   MeSH
• HEK293 buňky MeSH
• hydrofobní a hydrofilní interakce MeSH
• ivermektin chemie   farmakologie   MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• metoda terčíkového zámku MeSH
• molekulární modely MeSH
• mutageneze cílená MeSH
• purinergní receptory P2X4 chemie   genetika   metabolismus   MeSH
• sekundární struktura proteinů MeSH
• transfekce MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Intramural MeSH
• Názvy látek
• adenosintrifosfát MeSH
• aminokyseliny MeSH
• ivermektin MeSH
• purinergní receptory P2X4 MeSH