HİPOTALAMUS NÖRONLARINDA KALSİYUM SİNYALLEŞMESİ. Doç. Dr. Mete ÖZCAN Fırat Üniversitesi Tıp Fakültesi Biyofizik Anabilim Dalı

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1 HİPOTALAMUS NÖRONLARINDA KALSİYUM SİNYALLEŞMESİ Doç. Dr. Mete ÖZCAN Fırat Üniversitesi Tıp Fakültesi Biyofizik Anabilim Dalı

2 Kalsiyum; Çeşitli hücresel fizyolojik olayların düzenlenmesinde rol oynayan bir ikincil haberci dir. Uyarılabilme Ekzositozis Motilite Apoptozis Gen transkripsiyonu Hücre farklılaşması

4 Özel floresan problar kullanılarak canlı hücrelerdeki miktarı ölçülebilir. Kalsiyum İndikatörleri: BAPTA Fura-2 Fluo-4 Rhod-2 Electron-donating CH 3 or electron-withdrawing NO 2 groups increase affinity for Ca 2+

8 GT1-7 Nöronları: Effects of subtype-specific Ca2+ channel blockers on the peak Ca2+ currents of GT1 7 cells. Watanabe M et al. Endocrinology 2004;145: by Endocrine Society

9 Role of Metastin in the Release of Gonadotropin Releasing Hormone From the Hypothalamus of the Male Rat Journal of Andrology Volume 27, Issue 3, pages , 2 JAN 2013 DOI: /jandrol

27, Issue 3, pages 444-449, 2 JAN 2013 DOI: 10.2164/jandrol.

10 Kisspeptin depolarizes GnRH neurons. Liu X et al. Endocrinology 2008;149: by Endocrine Society

11 Ba2+ and FFA decrease the number of kisspeptin-responsive GnRH neurons. Liu X et al. Endocrinology 2008;149: by Endocrine Society

12 Fig. 1 Calcium signals activated by kisspeptin-10 in GT1-7 cells. (A) Kisspeptin-10 induces long-lasting intracellular calcium elevation in GT1-7 cells. (B) Bar graph compares effects of different concentrations of kisspeptin-10 on percentage of fluorescen... Mete Ozcan, Ergul Alcin, Ahmet Ayar, Bayram Yilmaz, Suleyman Sandal, Haluk Kelestimur Kisspeptin-10 elicits triphasic cytosolic calcium responses in immortalized GT1-7 GnRH neurones Neuroscience Letters Volume 492, Issue

(B) Bar graph compares effects of different concentrations of kisspeptin-10 on percentage of fluorescen.

13 Fig. 2 (A) PKC inhibitor, chelerythrine chloride, inhibited kisspeptin-10-induced [Ca 2+ ] <ce:italic> i</ce:italic> responses in GT1-7 cells. (B) Bar graph compares effects of kisspeptin-10 and kisspeptin-10<ce:hsp sp="0.25"/> +<ce:hsp sp="0.25"/> PKC i... Mete Ozcan, Ergul Alcin, Ahmet Ayar, Bayram Yilmaz, Suleyman Sandal, Haluk Kelestimur Kisspeptin-10 elicits triphasic cytosolic calcium responses in immortalized GT1-7 GnRH neurones Neuroscience Letters Volume 492, Issue

.. Mete Ozcan, Ergul Alcin, Ahmet Ayar, Bayram Yilmaz, Suleyman Sandal, Haluk Kelestimur Kisspeptin-10 elicits triphasic cytosolic

14 Kroll H, Bolsover S, Hsu J, Kim SH, Bouloux PM. Kisspeptin-evoked calcium signals in isolated primary rat gonadotropin- releasing hormone neurones. Neuroendocrinology. 2011;93(2):

15 Bazal GnIH (1µM) 1,8 Floresan Oraný (340nm/380nm) 1,6 1,4 1,2 1,0 0,8 0,6 * 0,0 Bazal GnIH GnIH+Kisspeptin GnIH (1µM)+Kisspeptin (100nM) Ortalama Veriler(n=34, *p<0.001) Şekil. GT1-7 Hücrelerinde GnIH ve GnIH+kisspeptinin kalsiyum sinyali üzerine etkileri (n=34).

(100nM) Ortalama Veriler(n=34, *p<0.001) Şekil.

16 Kelestimur H, Kacar E, Uzun A, Ozcan M, Kutlu S. Arg-Phe-amide-related peptides influence gonadotropin-releasing hormone neurons. Neural Regen Res Jun 25;8(18):

19 Fig.?1 Melatonin provokes calcium elevation in GT1?7 cells in a dose-dependent manner. 1<ce:hsp sp="0.25"/>?m (n<ce:hsp sp="0.12"/> =<ce:hsp sp="0.12"/> 25), 10<ce:hsp sp="0.25"/>?m (n<ce:hsp sp="0.12"/> =<ce:hsp sp="0.12"/> 68) and 100<ce:hsp sp="0.25"/... Haluk Kelestimur, Mete Ozcan, Emine Kacar, Ergul Alcin, Bayram Yilmaz, Ahmet Ayar Melatonin elicits protein kinase C-mediated calcium response in immortalized GT1?7 GnRH neurons Brain Research Volume

?m (n<ce:hsp sp="0.12"/> =<ce:hsp sp="0.12"/> 68) and 100<ce:hsp sp="0.25"/.

20 Fig.?3 Summary of the effects of (A) melatonin receptor antagonist luzindole, (B) PKC inhibitor, chelerythrine chloride (ChCl) on melatonin fluorescence calcium responses in GT1?7 cells. (C) Cells were stimulated by melatonin (100<ce:hsp sp="0.25"/>?m) an... Haluk Kelestimur, Mete Ozcan, Emine Kacar, Ergul Alcin, Bayram Yilmaz, Ahmet Ayar Melatonin elicits protein kinase C-mediated calcium response in immortalized GT1?7 GnRH neurons Brain Research Volume

.. Haluk Kelestimur, Mete Ozcan, Emine Kacar, Ergul Alcin, Bayram Yilmaz, Ahmet Ayar Melatonin elicits protein kinase C-mediated

21 Sasson R, Dearth RK, White RS, Chappell PE, Mellon PL. Orexin A induces GnRH gene expression and secretion from GT1-7 hypothalamic GnRH neurons. Neuroendocrinology. 2006;84(6):

22 1,4 p<0.01 n=15 1,3 1,2 Bazal Orexin (1µM) Floresan Oraný 1,1 1,0 0,9 0,8 0,0 Bazal 1uM ORX A Bazal Orexin (1µM)

23 In vivo leptin alters the postsynaptic response of GnRH neurons Sullivan S D et al. J. Neurosci. 2003;23: by Society for Neuroscience

24 1,0 0,9 Floresan Oraný (340 nm/380 nm) 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0,0 Bazal Leptin Şekil. GT1-7 Hücrelerinde leptinin kalsiyum sinyali üzerindeki etkileri

25 Fig. 1 Immortalized, clonal cell lines exhibit unique cellular morphologies, and neuroendocrine markers. (A) Representative phasecontrast micrographs of clonal cell lines, rhypoe-7 and rhypoe-8. (B, C) RT-PCR of embryonic rat hypothalamic cell lines. Rat... S. Gingerich, X. Wang, P.K.P. Lee, S.S. Dhillon, J.A. Chalmers, M.M. Koletar, D.D. Belsham The generation of an array of clonal, immortalized cell models from the rat hypothalamus: analysis of melatonin effects on kisspeptin and gonadotropin-inhibitory hormone neurons Neuroscience, Volume 162, Issue 4, 2009,

26 Fig. 2 Characterization of pertinent cellular markers in rhypoe-xx cell lines. RT-PCR was performed on the 32 rat cell lines for the indicated cellular markers. Rat hypothalamic RNA was used as a positive control, whereas reactions without template were al... S. Gingerich, X. Wang, P.K.P. Lee, S.S. Dhillon, J.A. Chalmers, M.M. Koletar, D.D. Belsham The generation of an array of clonal, immortalized cell models from the rat hypothalamus: analysis of melatonin effects on kisspeptin and gonadotropin-inhibitory hormone neurons Neuroscience, Volume 162, Issue 4, 2009,

27 Leptin caused a dose-dependent increase in [Ca 2+ ] i in all subtypes of rhypo-7 neurons Fig. 1 Dose-dependent effects of leptin on intracellular calcium levels in immortalized RFamide-related peptide-3 neurons. (A) 1 µm (n=28) and 10 µm (n=49) leptin evoked Ca 2+ responses in different rhypo-7 cells, in the presence of extracellular Ca 2+, Mete Ozcan, Tugrul Saatci, Ahmet Ayar, Sinan Canpolat, Haluk Kelestimur Leptin activates cytosolic calcium responses through protein kinase-c dependent mechanism in immortalized RFamiderelated peptide-3 neurons Brain Research, Volume 1601, 2015,

28 In the presence of a Ca 2+ -free extracellular solution, leptin showed weaker effect on intracellular calcium concentration Fig. 2 Effects of leptin (10 µm) on intracellular calcium levels of rhypo-7 neurons in calcium-free bathing conditions. (A) Original representative trace and (B) Bar graph show that the intracellular calcium response of a rhypo-7 cell to acute perfusion of... Mete Ozcan, Tugrul Saatci, Ahmet Ayar, Sinan Canpolat, Haluk Kelestimur Leptin activates cytosolic calcium responses through protein kinase-c dependent mechanism in immortalized RFamiderelated peptide-3 neurons Brain Research, Volume 1601, 2015,

29 PKC inhibition with chelerythrine chloride (ChCl) abolishes the increase in intracellular calcium concentration due to leptin Fig. 3 Effects of PKC inhibitor chelerythrine chloride (ChCl) on leptin-induced intracellular calcium responses in rhypo-7 neurons. Values are given as a percentage of the oxytocin induced fluorescence (340/380 nm) ratio and expressed as mean±sd from three... Mete Ozcan, Tugrul Saatci, Ahmet Ayar, Sinan Canpolat, Haluk Kelestimur Leptin activates cytosolic calcium responses through protein kinase-c dependent mechanism in immortalized RFamiderelated peptide-3 neurons Brain Research, Volume 1601, 2015,

30 Fig. 4 Effects of leptin on gonadotropin-inhibitory hormone secretion in rhypo-7 cells. Box graph compares the effects of leptin on gonadotropin-inhibitory hormone secretion at different time (15, 30 and 60 min) n =7, each group (on... Mete Ozcan, Tugrul Saatci, Ahmet Ayar, Sinan Canpolat, Haluk Kelestimur Leptin activates cytosolic calcium responses through protein kinase-c dependent mechanism in immortalized RFamiderelated peptide-3 neurons Brain Research, Volume 1601, 2015,

31 Possible action mechanism of leptin on RFRP-3 neurons LEP Ca 2+ LEPR Inositol Phospholipid Diacylglycerol VGCC? PLC P PKC P P Inositol 1,4,5 triphosphate (IP 3 ) P P P Ca 2+ Endoplasmic Reticulum

32 Adapted from Tena-Sempere M Eur J Endocrinol 2012;167:

33 GnIH effects on HypoE-8 cells

34 RFRP-1 (1µM)+ Kisspeptin (100nM) Basal RFRP -1(1µM) 1,6 * Floresan Oraný (340nm/380nm) 1,4 1,2 1,0 0,8 0,6 0,0 Bazal RFRP RFRP+Kisspeptin

35 Basal GnIH (1µM) GnIH (1µM)+ Kisspeptin (100nM) HiKCl +

36 Bazal Kisspeptin RFRP (1µM)+ Kisspeptin (100nM) GnIH (1µM)+ Kisspeptin (100nM)

37 TEŞEKKÜRLER

40 Intracellular messengers involved in the generation of calcium signalling IP3, AA, cadpr

41 An interesting biological model for the study of the complex interplaying among calcium and intracellular messengers (AA, NO and others): endothelial cells

46 Floresan boyaların yaydığı emisyon, ortamdaki değişikliklerden etkilenmektedir. Kalsiyum Sodyum Magnezyum ph değişiklikleri spesifik floroforun yaydığı emisyonu etkiler. Bu sayede uygun floresan boya seçilerek hücre içi iyon konsantrasyonu değişiklikleri gerçek zamanlı tespit edilebilir.

48 Problemler 1. Diğer maddelerle (formaldehyde ve acetic acid) etkileşimi toksik potansiyele sebep olabilir. 2. Bölümlere ayırmak: Hücre içerisindeki yapılarda AM esters birikir Polianyonik formdaki indikatörler aktif taşımayla organellerin içinde ayrılabilir 3. AM ester hidrolizisinin tamamlanamaması: Belirli bir kısmı hidrolize olmuş AM esters Ca +2 duyarsızdır, buda Ca 2+ deteksiyonunu zorlaştırır. 4. Sızıntı: organik iyon transportu ile hücrelerden anyonik indikatörlerin çıkması fura-2 AM ester (Oksijenli asitler ile alkollerin aralarından bir su molekülü ayrılması sonucunda ortaya çıkan madde)

50 Patch Kenetleme Tekniği

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