Kemirgen Hipotalamusunda Glutamatın Atriyal Natriüretik Peptit Nöronları Üzerindeki Etkilerinin İmmünohistokimyasal Yöntemle Araştırılması

Year 2020, Volume: 39 Issue: 2, 73 - 81, 30.12.2020

Duygu Gök Yurtseven , Zehra Minbay , Özhan Eyigör

https://doi.org/10.30782/jrvm.807799

Abstract

Atriyal natriüretik peptit (ANP) organizmanın sıvı homeostazında önemli etkileri olan, özellikle de kan basıncı ile sıvı elektrolit dengesinin düzenlenmesinde rol oynayan bir peptittir. Yoğun olarak atrial kalp kası hücrelerince üretilen ANP, merkezi sinir siteminde (MSS) lokalize bir grup nöron tarafından da eksprese edilir. MSS’de nöronal ağlarda impuls iletiminde çok önemli rolü olan glutamaterjik sistemin ANP nöronlarını kontrol eden etkileri ile ilgili bilgi literatürde yer almamaktadır. Sunulan çalışma kapsamında, hipotalamusun supraoptik çekirdeğinde lokalize ANP nöronları üzerinde glutamat agonistlerinin etkileri ve bu nöronlarda eksprese olan glutamat reseptör alt birimlerinin varlığı, ikili immünoperoksidaz ve immünoflouresans yöntemler kullanılarak araştırılmıştır. Glutamat agonistlerinin etkilerini belirlemek üzere, kainik asit, AMPA ve NMDA, kontrol grupları için salin, antagonist olarak CNQX ve MK801 enjeksiyonu yapılan sıçanları içeren deney grupları oluşturulmuştur. ANP nöronlarındaki glutamaterjik innervasyonun belirlenmesinde veziküler glutamat transporter (VGluT) proteinleri ve nöronal aktivasyonun gösterilmesinde ise c-Fos immünoreaktivitesinin varlığı belirteç olarak kullanılmıştır. Çalışmaların sonucunda; glutamat agonistlerinin ANP nöronlarında nöronal aktivasyonu anlamlı bir şekilde arttırdığı, bu artışın antagonistler ile anlamlı bir şekilde baskılanabildiği belirlenmiştir. Ayrıca ANP nöronlarının glutamat reseptörlerine ait alt birimlerden GluA1, GluA2, GluK1/2/3 ve GluK5’i eksprese ettikleri ve VGluT içeren glutamaterjik akson sonlanmalarıyla temasta oldukları saptanmıştır. Sonuç olarak, bu çalışma ile glutamatın ANP nöronlarında aktive edici etkiye sahip olduğu ve glutamat reseptörlerinin bu nöronlarca eksprese edildiği, dolayısıyla da bu nöronların glutamaterjik uyarıları alabilecek mekanizmaya sahip oldukları gösterilmiştir. Bu sonuçlar, ANP nöronlarının merkezi düzenlenmesinde glutamaterjik sistemin önemli bir rol oynayabileceğini düşündürmüştür.

Keywords

Atrial natriüretik peptit, Glutamat, Kainik Asit, c-Fos, VGluT, Sıçan

Supporting Institution

TÜBİTAK

Project Number

104S286

Thanks

Bu makalede yer alan laboratuvarımıza ait sonuçlar, TÜBİTAK tarafından desteklenen 104S286 nolu proje kapsamında yapılan çalışmalardan elde edilmiştir.

Investigation of Glutamatergic Effects on Atrial Natriuretic Peptide Neurons by Immunohistochemical Method in Rodent Hypothalamus

Abstract

Atrial natriuretic peptide (ANP) is a peptide that has important effects on the fluid homeostasis of the organism, particularly involved in the regulation of blood pressure and fluid electrolyte balance. ANP, which is intensely produced in the heart atrium, is also expressed in different peripheral and central organs. There is no knowledge in the literature regarding the control of ANP neurons in the central nervous system by the glutamatergic system. In our study, the effects of glutamate agonists on ANP neurons and the presence of glutamate receptor subunits expressed in these neurons were investigated using dual immunoperoxidase and immunofluorescence methods. In the present study, ANP neurons localized in the supraoptic nucleus (SON) of the hypothalamus were examined. In order to determine the effects of glutamate agonists, experimental groups were formed containing rats which were injected with kainic acid, AMPA and NMDA, saline for control groups and CNQX or MK801 as antagonists. VGluT protein was used as a marker for the determination of glutamatergic innervations in ANPergic neurons, and c-Fos immunoreactivity was used as a marker for neuronal activation. It has been determined that glutamate agonists cause activation in a significant number of ANP neurons which can significantly be suppressed by antagonists. In addition, it was determined that the ANP neurons express GluA1, GluA2, GluK5 and GluK1/2/3, which are subunits of glutamate receptors, and are in contact with glutamatergic axon terminations containing VGluT. As a result, it has been shown that glutamate has an effect on the activation of ANP neurons and that glutamate receptors are expressed by these neurons, thus, these neurons possess the mechanism in order to receive glutamatergic stimulations. In conclusion, these results suggested that the glutamatergic system plays an important role in the central regulation of ANP neurons.

Keywords

Atrial natriüretic peptid, Glutamate, Kainic Acid, c-Fos, VGluT, Rat

Project Number

104S286

References

• 1. de Bold AJ, Borenstein HB, Veress AT, et al. A rapid and potent natriuretic response to intravenous injection of atrial myocardial extract in rats. Life Sci. 1981;28(1):89-94.
• 2. Sudoh T, Kangawa K, Minamino N, et al. A new natriuretic peptide in porcine brain. Nature 1988;332:78-81.
• 3. Sudoh T, Minamino N, Kangawa K, et al. C-type natriuretic peptide (CNP): a new member of natriuretic peptide family identified in porcine brain. Biochem. Biophys. Res. Commun. 1990;168(2):863-70.
• 4. Flynn, TG, de Bold ML, de Bold AJ. The amino acid sequence of an atrial peptide with potent diuretic and natriuretic properties. Biochem. Biophys. Res. Commun. 1983;117(3):859-65.
• 5. Hodes A, Lichtstein D. Natriuretic hormones in brain function. Front. Endocrinol. 2014;5:1-13.
• 6. Edwards BS, Zimmerman RS, Schwab TR, et al. Atrial stretch, not pressure, is the principal determinant controlling the acute release of atrial natriuretic factor. Circ. Res. 1988;62(2):191-195.
• 7. Tanaka I, Misono KS, Inagami T. Atrial natriuretic factor in rat hypothalamus, atria and plasma: determination by specific radioimmunoassay. Biochem. Biophys. Res. Commun. 1984;124(2):663-668.
• 8. Saavedra JM. Regulation of atrial natriuretic peptide receptors in the rat brain. Cell Mol Neurobiol 1987;7(2):151-173.
• 9. Chriguera RS, Rocha MJA, Antunes-Rodriguesa J, et al. Hypothalamic atrial natriuretic peptide and secretion of oxytocin. Brain Res. 2001;889(1-2):239-242.
• 10. Palkovits M, Eskay RL, Antoni FA. Atrial natriuretic peptide in the median eminence is of paraventricular nucleus origin. Neuroendocrinology 1987;46(6):542-544.
• 11. Johnson AK. The periventricular anteroventral third ventricle (AV3V): its relationship with the subfornical organ and neural systems involved in maintaining body fluid homeostasis. Brain Res. Bull. 1985;15(6):595-601.
• 12. Kawata M, Ueda S, Nakao K, et al. Immunohistochemical demonstration of alpha-atrial natriuretic containing neurons in the rat brain. Histochemistry 1985;83(1):1-3.
• 13. Saper CB, Hurley KM, Moga MM, et al. Brain natriuretic peptides: Differential localization of a new family of neuropeptides. Neurosci. Lett. 1989;96(1);29-34.
• 14. Farina LE, Lipari D, Dieli F, et al. Atrial natriuretic peptide secretion during development of the rat supraoptic nucleus. Eur. J. Histochem. 2005;49(4):379-384.
• 15. Lipari EF, Lipari A, Dieli F, et al. ANP presence in the hypothalamic suprachiasmatic nucleus of developing rat. Ital. J. Anat. Embryol. 2007;112(1):19-25.
• 16. Brann DW, Mahesh VB. Excitatory amino acids: function and significance in reproduction and neuroendocrine regulation. Front. Neuroendocrinol. 1994;15(1):3-49.
• 17. Brann DW, Zamorano PL, Chorich LP, et al. Steroid hormone effects on NMDA receptor binding and NMDA receptor mRNA levels in the hypothalamus and cerebral cortex of the adult rat. Neuroendocrinology 1993;58(6):666-672.
• 18. Gereau RW, Swanson GT, eds. The Glutamate Receptors. 1st ed. Totowa NJ: Humana Press; 2008.
• 19. Hollmann M, Heinemann S. Cloned glutamate receptors. Annu. Rev. Neurosci. 1994;17:31-108.
• 20. Bettler B, Mulle C. Neurotransmitter receptors II. AMPA and kainate receptors. Neuropharmacology 1995;34(2):123-139.
• 21. Mori H, Mishina M. Structure and function of the NMDA receptor channel. Neuropharmacology 1995;34(10):1219-1237.
• 22. Kew JN, Kemp JA. Ionotropic and metabotropic glutamate receptor structure and pharmacology. Psychopharmacology 2005;179(1):4-29.
• 23. Niciu MJ, Kelmendi B, Sanacora G. Overview of glutamatergic neurotransmission in the nervous system. Pharmacol Biochem Behav 2012;100(4):656-64.
• 24. Sagar SM, Sharp FR, Curran T. Expression of c-Fos protein in brain: metabolic mapping at the cellular level. Science. 1988;240(4857):1328-1331.
• 25. Hoffman GE, Smith MS, Verbalis JG. c-Fos and related immediate early gene products as markers of activity in neuroendocrine systems. Front. Neuroendocrinol. 1993;14(3):173-213.
• 26. Eriksson M, Ceccatelli S, Uvnäs-Moberg K, et al. Expression of Fos-related antigens, oxytocin, dynorphin and galanin in the paraventricular and supraoptic nuclei of lactating rats. Neuroendocrinology 1996;63:356-367.
• 27. Paxinos G, Watson C, eds. The rat brain in stereotaxic coordinates. 6th edition, Elsevier Academic Press, Amsterdam; 2009.
• 28. Gutkowska J, Antunes-Rodrigues J, McCann SM. Atrial natriuretic peptide in the brain and pituitary gland. Physiol. Rev. 1997;77(2):465-515.
• 29. McCann SM, Antunes-Rodrigues J, Jankowski M, et al. Oxytocin, vasopressin and atrial natriuretic peptide control body fluid homeostasis by action on their receptors in brain, cardiovascular system and kidney. Prog. Brain Res. 2002;139:309-328.
• 30. Chriguer RS, Rocha MJ, Antunes-Rodrigues J, et al. Hypothalamic atrial natriuretic peptide and secretion of oxytocin. Brain Res. 2001;889(1-2):239-242.
• 31. Gutkowska J, Antunes-Rodrigues J, McCann SM. Atrial natriuretic peptide in brain and pituitary gland. Physiol. Rev. 1997;77(2):465-515.
• 32. Lauand F, Ruginsk SG, Rodrigues HLP, et al. Glucocorticoid modulation of atrial natriuretic peptide, oxytocin, vasopressin and Fos expression in response to osmotic, angiotensinergic and cholinergic stimulation. Neuroscience 2007;147(1):247-57.
• 33. Ku YH, Zhang T. Brain atriopeptin mediates AV3V depressor response. Peptides 1994;15(6):1053-1056.
• 34. Ku YH, Li YH. Inhibitory effect of atriopeptinergic neurons in AV3V region on AngiotensinII pressor system in rat brain. Peptides 2004;25(4):615-620.
• 35. Hoffman GE, Smith MS, Verbalis JG. c-Fos and related immediate early gene products as markers of activity in neuroendocrine systems. Front. Neuroendocrinol 1993;14(3):173-213.
• 36. Minbay FZ, Eyigor O, Cavusoglu I. Kainic acid activates oxytocinergic neurons through non-NMDA glutamate receptors. Int. J. Neurosci. 2006;116(5):587-600.
• 37. Eyigor O, Minbay Z, Cavusoglu I. Activation of orexin neurons through non-NMDA glutamate receptors evidenced by c-Fos immunohistochemistry. Endocrine 2010;37(1):167-72.
• 38. Eyigor O, Minbay Z, Kafa IM. Glutamate and Orexin Neurons. Vitam. Horm: Sleep Hormones 2012;89:209-222.
• 39. Serter Kocoglu S, Gok Yurtseven D, Minbay FZ, et al. Glutamatergic activation of neuronostatin neurons in the periventricular nucleus of the hypothalamus. Brain Sci. 2020;10(4);217.
• 40. Gok Yurtseven D, Serter Kocoglu S, Minbay Z, et al. Immunohistochemical evidence for glutamatergic regulation of nesfatin-1 neurons in the rat hypothalamus. Brain Sci. 2020;10(9):630.
• 41. Csaki A, Kocsis K, Kiss J, et al. Localization of putative glutamatergic/aspartatergic neurons projecting to the supraoptic nucleus area of the rat hypothalamus. Eur. J. Neurosci. 2002; 16(1);55-68.
• 42. Malarkey EB, Parpura V. Mechanisms of glutamate release from astrocytes. Neurochem. Int. 2008;52(1-2):142-154.
• 43. Eyigor O, Centers A, Jennes L. Distribution of ionotropic glutamate receptor subunit mRNAs in the rat hypothalamus. J. Comp. Neurol. 2001;434(1):101-124.
• 44. van den Pol AN, Hermans-Borgmeyer I, Hofer M, et al. Ionotropic glutamatereceptor gene expression in hypothalamus: localization of AMPA kainate and NMDA receptor RNA with in situ hybridization. J. Comp. Neurol. 1994;343(3):428-44.
• 45. Monyer H, Sprengel R, Schoepfer R, et al. Heteromeric NMDA receptors: molecular and functional distinction of subtypes. Science 1992;256(5060):1217-1221.
• 46. Howe JR. Homomeric and heteromeric ion channels formed from the kainatetype subunits GluR6 and KA2 have very small, but different, unitary conductances. J. Neurophysiol.1996;76(1):510-519.
• 47. Alt A, Weiss B, Ogden AM, et al. Pharmacological characterization of glutamatergic agonists and antagonists at recombinant human homomeric and heteromeric kainate receptors in vitro. Neuropharmacology 2004;46(6):793-806.
• 48. Takamori S, Rhee JS, Rosenmund C, et al. Identification of a vesicular glutamate transporter that defines a glutamatergic phenotype in neurons. Nature 2000;407(6801):189-94.
• 49. Moriyama Y, Yamamoto A. Glutamatergic chemical transmission: Look! Here, There, and anywhere. J. Biochem. 2004;135(2):155-63.
• 50. Takamori S. VGLUTs: 'Exciting' Times for Glutamatergic Research?. Neurosci Res 2006; 55(4):343-351.
• 51. Liguz-Lecznar M, Skangiel-Kramska J. Vesicular Glutamate Transporters (VGLUTs): the Three Musketeers of Glutamatergic System. Acta Neurobiol. Exp. (Wars) 2007;67(3):207-18.
• 52. Kaneko T, Fujiyama F. Complementary distribution of vesicular glutamate transporters in the central nervous system. Neurosci. Res. 2002;42(4):243-250.
• 53. Kaneko T, Fujiyama F, Hioki H. Immunohistochemical localization of candidates for vesicular glutamate transporters in the rat brain. J. Comp. Neurol. 2002;444(1):39-62.
• 54. Hisano S, Nogami H. Transporters in the neurohypophysial neuroendocrine system, with special reference to vesicular glutamate transporters (BNPI and DNPI): a Review. Microsc. Res. Tech. 2002;56(2):122-131.