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