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

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

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

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