Immunhistochemische, zellbiologische und physiologische in vivo Untersuchungen zum Apelin/APJ-Systemin Hypothalamus und Hypophyse der Ratte

The hypothalamus represents the most important component of the central nervous system (CNS) used for integrating afferent signaling information and regulating the homeostasis of body temperature, water and electrolyte balance of the extracellular space, circadian rhythm and energy metabolism. The most important hypothalamic structure (nucleus) for regulating the above mentioned physiological functions is the Nucleus paraventricularis (PVN) of the preoptic region, which is located closely to the third brain ventricle. The PVN consists of many neurosecretory cells and shows numerous efferent and afferent neuronal connections to other structural components of the brain. In addition to the PVN, the Nucleus praeopticus medianus (MnPO), which is located close to the Commisura anterior, displays a critical integrative function for regulating water and electrolyte balance, as well as regulating body temperature. Besides other peptides and classical neurotransmitters, the gaseous transmitter nitric oxide (NO), produced by the neuronal nitric oxide synthase (nNOS) amongst other substances, plays an essential neuromodulatory role in regulating water and electrolyte balance and body temperature. The apelinergic system, consisting of several active fragments of the preproapelin (apelin36, apelin17 and apelin13) and the G-protein coupled receptor, APJ, has been reported to also centrally modulate body temperature and water and electrolyte balance. Expression of apelin peptides and their receptors has been shown in peripheral organs and the CNS, in particular in the hypothalamus. Despite many expression studies having been undertaken, the detailed mapping of the APJ protein distribution in hypothalamus and thalamus remains to be determined. (1) The present thesis therefore includes, for the first time, a detailed semi-quantitative immunhistochemical mapping of the APJ protein on coronal hypothalamic rat brain slices and confirms published APJ mRNA expression in structures like the PVN and Nucleus supraopticus (SON). However, for many structures and/or nuclei like the MnPO or the medial devision of the bed nucleus of the stria terminalis, the N. praecommisuralis and the N. dorsomedialis, APJ protein has been described for the first time in single neurons or pseudopodia. (2) The expression of nNOS has been reported in both the PVN and MnPO previously. Additionally it has been postulated that apelinergic effects, for example on the cardiovascular system, could be mediated by NO. Therefore, the present work analyzed the possible immunhistochemical codistribution and colocalization of APJ and nNOS in the hypothalamus. In this study the MnPO, amongst other brain structures, showed distinct codistribution of nitrergic neurons and APJ immunopositive fiber structures. Quantitative analysis of the PVN and SON perikarya revealed about 40 % and, 50% cellular colocalization of both molecules. Due to this prominent colocalization and codistribution in the PVN and MnPO, NO could be involved as a downstream signaling molecule in mediating central apelinergic functions. (3) Because of the distinctive expression of APJ in the MnPO and PVN, specificly the primary neuroglial cultures of both nuclei and additionally primary intermediate lobe pituitary (HZL) cultures, with high native expression of APJ, these cultures were used to analyze apelin induced intracellular signal transduction in vitro. Changes of intracellular calcium concentrations ([Ca2+]i), which are important second or third messengers, were monitored in single cells by calcium binding fluorophors fura-2 (=calcium imaging). Neurons and astrocytes of the MnPO and PVN specific cultures exhibited 2-9 % specific microglial cells in the MnPO and 12% specific microglial cells in the PVN. Of the MnPO and PVN cell cultures, 12 % directly responded with changes in ([Ca2+]i ) when stimulated with the pyroglutamate modified form of apelin13 (PyrAp13) (10 -6 mol/l). As described for other neuropeptides, PyrAp13 was able to positively and negatively modulate the influx of calcium caused by the classical neurotransmitter glutamate in mainly non-nitregic cells, but also nitregic cells. This modulatory function of PyrAp13 could provide evidence for the cellular basis of apelin as a novel CNS neuromodulator. Besides analysis of [Ca2+]I as a downstream signal of the apelinergic system, some reports showed involvement of extracellular regulated kinase (ERK)1/2 in apelinergic signaling in peripheral or transfected cell systems. In the present work no activation of ERK1/2 was detected in primary HZL cultures with highly native APJ expression, using Western Blot analysis. The lack of ERK1/2 expression may be due to already high levels of phosphorylation before stimulation. (4) As a next important component of the present work, in vivo parameters were monitored in rats by radiotelemetry after intracerebroventricular (i.c.v.) micro application of PyrAp13 (20 nmol). (A) Microinjection of PyrAp13 i.c.v. induced no significant changes in bacterial lipopolysaccharide (LPS)-induced sickness behavior, including anorexia, adipsia and lethargy. However, it significantly reduced LPS-induced fever 3-6 and 6-9 h after injection. Moreover, it also led to a significant reduction in plasma levels of tumor necrosis factor- α (TNF-α), a prominent proinflammatory cytokine, 2 h after injection. (B) Despite a high sequence of similarity between the APJ and the Angiotensin II (AngII) receptor and a proven interaction of both in vitro, i.c.v. microinjected PyrAp13 exhibited no influence on water intake in vivo or nuclear translocation of the transcription factor and neuronal activity marker c-Fos, induced by i.c.v. AngII. (C) Furthermore i.c.v. PyrAp13 showed no influence on telemetrically recorded parameters, such as body temperature, locomotor activity and food/water intake of animals.