Stimulation of AP evoke release of Ang II in the NTS detected by R‐GECO AT1R sniffer cells

Abstract

The renin angiotensin system (RAS) signaling in the brain plays an important role in cardiovascular control and body fluid homeostasis. Although systemic angiotensin II (Ang II) can access the brain through circumventricular organs such as the subfornical organ (SFO), organum vaculosum of the lamina terminalis (OVLT) and area postrema (AP), the existence of a brain RAS is still controversial. Our previous studies have used angiotensin sensitive sniffer cells to test whether angiotensin peptides are released from brain slices containing the SFO projections to the median preoptic nucleus. Sniffer cells were produced by transfecting Chinese Hamster Ovary cells with commercially available plasmids for the angiotensin type 1a (AT1a) receptor (Origene Tech.) and R‐GECO (Addgene #32462). These sniffer cells are sensitive to angiotensin II and III but not angiotensin 1–7, bradykinin, or neurotransmitters such as glutamate or acetylcholine. In these studies, we examined another pathway involving the AP and nucleus of tractus solitarius (NTS). The AP is angiotensin sensitive and projects to areas in the central nervous system such as the dorsal motor nucleus of the vagus, ventrolateral medulla, and NTS known to be important areas in autonomic regulation of cardiovascular function. The purpose of this study was to test for the release of angiotensin peptides in the NTS after stimulation of AP. Coronal brainstem slices (300 um) containing both the AP and NTS were prepared from adult male Sprague – Dawley rats that were anesthetized with isoflurane (2–3%). The slices were perfused with oxygenated artificial cerebrospinal fluid (containing containing (in mM): 126 NaCl, 3.0 KCl, 2.0 CaCl2, 2.0 MgSO4, 1.25 NaH2PO4, 26 NaHCO3, 10 and D‐Glucose; 300 mOsm, pH 7.4). Sniffer cells were placed on the NTS and imaged on an Olympus B51WI equipped with epifluorescence using CellSens Dimensions software (Olympus). Sniffer cell images were captured once every second. Changes in fluorescent intensity of sniffer cells in the NTS was determined following electric stimulation of the AP (100Hz, 10ms, 1mA). Electrical stimulation increased fluorescence intensity 134 ± 11%, n=13 of sniffer cells on the NTS with a mean response latency of 4 ± 0.7sec, n=13. Some cells demonstrated spontaneous changes in fluorescence intensity 2±0.1, n=28 that were not observed in cells located outside of the NTS. The results indicate that sniffer cells placed on the NTS demonstrated evidence of spontaneous and evoked release of angiotensin peptides.Support or Funding InformationP01 HL088052