Molekulární mechanismy a význam purinergní signalizace v hypotalamu

(CZ): Extracelulární ATP a purinerginí P2 receptory (P2X a P2Y) hrají roli v signalizačním systému zvaném "purinergní signalizace", který je hojně využíván jak somatickýmí tak nervovými tkáněmi, a působí také v endokrinním systému. Středem zájmu mé disertační práce je úloha a výskyt P2X a P2Y receptorů v hypotalamických supraoptických jádrech (SON) produkujících hormony vasopresin a oxytocin, a v suprachiasmatických jádrech (SCN), která představují centrum biologických hodin u savců. V první části mé práce jsme testovali hypotézu, že P2X a P2Y receptory se účastní zvýšené tvorby a sekrece hormonů v magnocelulárních SON neuronech stimulovaných prostřednictvím experimentálního protokolu hladovění/ dokrmování. Studovali jsme účinek 2 hodinového dokrmování po 48 hodinovém hladovění na expresi hormonální, P2X a P2Y mRNA v SON tkáni u 30ti denních potkanů, a účinek aplikace ATP na elektrofyziologické vlastnosti SON neuronů v mozkových řezech kontrolního a experimentálního potkana. Kvantitativní real-time PCR analýza ukázala, že exprese mRNA pro P2X2 a arginin vasopresin (AVP) je zvýšena a exprese P2Y1 mRNA snížena u potkanů vystavených hladovění/dokrmování ve srovnání s kontrolami, zatímco hladiny mRNA pro P2X4, P2X7, P2Y2 a oxytocin nebyly významně změněny. Snímání z celé buňky technikou patch clamp...(EN): Extracellular ATP and purinergic P2 receptors (P2X and P2Y) are involved in a signaling network called "purinergic signaling" which is widely exploited in both somatic and neuronal tissues, and is also operative in endocrine system. The main focus of my thesis is on the role and expression of P2X and P2Y receptors in hypothalamic supraoptic nuclei (SON) producing hormones vasopressin and oxytocin, and the suprachiasmatic nuclei (SCN), the principal circadian pacemaker in mammals. In the first part of my thesis, we tested the hypothesis that P2X and P2Y receptors play a role in the enhanced release of hormones from magnocellular SON neurons stimulated through fasting/refeeding experimental protocol. We studied the effect of 2 h of refeeding after 48 h of fasting on hormone, P2X and P2Y mRNA expression in the SON tissue of 30-day-old rats, and the effect of ATP on electrophysiological properties of SON neurons in brain slices from control and fasted/refed rats. Quantitative real-time PCR revealed that the expression of P2X2 and arginine-vasopressin (AVP) mRNA was increased and P2Y1 mRNA expression was decreased in fasted/refed rats compared to controls, whereas P2X4, P2X7, P2Y2 and oxytocin mRNA levels were not significantly changed. Whole-cell patch clamp recordings showed that the amplitude...Katedra fyziologieDepartment of PhysiologyPřírodovědecká fakultaFaculty of Scienc

The molecular mechanisms and role of purinergic signaling in hypothalamus

(EN): Extracellular ATP and purinergic P2 receptors (P2X and P2Y) are involved in a signaling network called "purinergic signaling" which is widely exploited in both somatic and neuronal tissues, and is also operative in endocrine system. The main focus of my thesis is on the role and expression of P2X and P2Y receptors in hypothalamic supraoptic nuclei (SON) producing hormones vasopressin and oxytocin, and the suprachiasmatic nuclei (SCN), the principal circadian pacemaker in mammals. In the first part of my thesis, we tested the hypothesis that P2X and P2Y receptors play a role in the enhanced release of hormones from magnocellular SON neurons stimulated through fasting/refeeding experimental protocol. We studied the effect of 2 h of refeeding after 48 h of fasting on hormone, P2X and P2Y mRNA expression in the SON tissue of 30-day-old rats, and the effect of ATP on electrophysiological properties of SON neurons in brain slices from control and fasted/refed rats. Quantitative real-time PCR revealed that the expression of P2X2 and arginine-vasopressin (AVP) mRNA was increased and P2Y1 mRNA expression was decreased in fasted/refed rats compared to controls, whereas P2X4, P2X7, P2Y2 and oxytocin mRNA levels were not significantly changed. Whole-cell patch clamp recordings showed that the amplitude..

Circadian ATP Release in Organotypic Cultures of the Rat Suprachiasmatic Nucleus Is Dependent on P2X7 and P2Y Receptors

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

Iron (fe) content in vegetation cover of the natural protected area Kosmaj, Serbia

This paper analyzes the iron (Fe) content of the vegetation at the beginning of the vegetative cycle for the period 2012/2013. Heavy metal content in the vegetative parts of ten plants (eight types of woody plants and two types of herbs) was analyzed in four locations. Iron (Fe) is a biogenic element present in plants in relatively small quantities. However, its ability to produce chelates, as well as being polyvalent, assorts it into a group of elements which have a significant impact on many physiological and biochemical processes. The analysis of iron content in leaves of woody plants and herbs growing in the natural protected area "Kosmaj" has shown that depending on location the iron concentration, in statistical terms, significantly differs and ranges from A to I in locations 1 and 3, whereas its concentration ranges from A to J in locations 2 and 4

Image1.pdf

<p>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.</p

Image4.pdf

<p>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.</p

Image2.pdf

<p>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.</p

Image3.pdf

<p>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.</p