Up‐regulation of hypothalamic arginine vasopressin by peripherally administered furosemide in transgenic rats expressing arginine vasopressin‐enhanced green fluorescent protein

Abstract

Furosemide, which is used worldwide as a diuretic agent, inhibits sodium reabsorption in the Henle's loop, resulting in diuresis and natriuresis. Arginine vasopressin (AVP) is synthesized in the supraoptic nucleus (SON), paraventricular nucleus (PVN), and suprachiasmatic nucleus (SCN) of the hypothalamus. The synthesis AVP in the magnocellular neurons of SON and PVN physiologically regulated by plasma osmolality and blood volume and contributed water homeostasis by increasing water reabsorption in the collecting duct. Central AVP dynamics after peripheral administration of furosemide remain unclear. Here, we studied the effects of intraperitoneal (i.p.) administration of furosemide (20 mg/kg) on hypothalamic AVP by using transgenic rats expressing AVP-enhanced green fluorescent protein (eGFP) under the AVP promoter. The i.p. administration of furosemide did not affect plasma osmolality in the present study; however, eGFP in the SON and magnocellular divisions of the PVN (mPVN) were significantly increased after furosemide administration compared to the control. Immunohistochemical analysis revealed Fos-like immunoreactivity (IR) in eGFP-positive neurons in the SON and mPVN 90 min after i.p. administration of furosemide, and AVP heteronuclear (hn) RNA and eGFP mRNA levels were significantly increased. These furosemide-induced changes were not observed in the suprachiasmatic AVP neurons. Furthermore, furosemide induced a remarkable increase in Fos-IR in the organum vasculosum laminae terminals (OVLT), median preoptic nucleus (MnPO), subfornical organ (SFO), locus coeruleus (LC), nucleus of the solitary tract (NTS), and rostral ventrolateral medulla (RVLM) after i.p. administration of furosemide. In conclusion, we were able to visualize and quantitatively evaluate AVP-eGFP synthesis and neuronal activations after peripheral administration of furosemide, using the AVP-eGFP transgenic rats. The results of this study may provide new insights into the elucidation of physiological mechanisms underlying body fluid homeostasis induced by furosemide. This article is protected by copyright. All rights reserved.

Oxytocin‐monomeric red fluorescent protein 1 synthesis in the hypothalamus under osmotic challenge and acute hypovolemia in a transgenic rat line

“…All rats were deeply sedated after administration of the anesthetic mixture, which consisted of medetomidine (3 mg/100 g), midazolam (4 mg/100 g), and butorphanol (5 mg/100 kg), before perfusion (Kawai et al, 2011;Ueno et al, 2019). Transcardial perfusion was performed as described previously (Ueno et al, 2018(Ueno et al, , 2019. After thoracotomy, the heart was carefully exposed.…”

“…Using the first group of sections, mRFP1 fluorescence in the hypothalamus was observed and photographed using a fluorescence microscope (ECLIPSE Ti-E; Nikon, Tokyo, Japan) with an RFP filter (Nikon). The obtained image was analyzed by an image analysis system (NIS-Elements; Nikon), as described in our previous reports (Ueno et al, 2018). The PVN and SON in the hypothalamus were identified with reference to a rat atlas (Paxinos & Watson, 2013…”

“…A fluorescent protein was used to label c-Fos protein using the immunohistochemical staining method described in previous reports (Motojima et al, 2016;Ueno et al, 2018Ueno et al, , 2019. The second group of sections mentioned above was incubated for 5 days at 4°C with a primary anti-Fos antibody solution (#sc-52; rabbit, Santa Cruz Biotechnology, Dallas, TX, USA; 1:500 in phosphate-buffered saline [PBS]).…”

“…To measure the changes of mRFP1 and OXT gene expressions in the PVN and SON for analysis by in situ hybridization histochemistry, transgenic rats were sacrificed by decapitation at 3 and 6 hr after administration of saline, HTN, and PEG at 10:00-12:00 (n = 6 in each group). In situ hybridization in this study was performed based on previous reports (Ueno et al, 2018(Ueno et al, , 2019Ueta, Levy, et al, 1995). Thus, only a brief explanation of the procedure of in situ hybridization is provided in this article.…”

“…The 35 S-labeled oligodeoxynucleotide probe and protocol for in situ hybridization histochemistry (ISH) in this experiment have been used many times before, and its reliability is considered guaranteed. The details of the probe sequence and the protocol for ISH have been described previously (Ueno et al, 2018(Ueno et al, , 2019Ueta, Levy, et al, 1995). The hybridized sections were exposed to autoradiography films (Amersham Hyperfilm, Buckinghamshire, UK) for 3 days (mRFP1) and 6 hr (OXT).…”