Regulation of the neuronal norepinephrine transporter by endothelin-1 and -3 in the rat anterior and posterior hypothalamus

Abstract

We previously reported that endothelin-1 and endothelin-3 modulate norepinephrine neuronal release and tyrosine hydroxylase activity and expression in the hypothalamus. In the present study we sought to establish the role of endothelin-1 and -3 in the regulation of norepinephrine uptake in the anterior and posterior hypothalamus. Results showed that in the anterior hypothalamus endothelin-3 increased neuronal norepinephrine uptake whereas endothelin-1 decreased it. Conversely, in the posterior hypothalamic region both endothelins diminished the neuronal uptake of the amine. Endothelins response was concentration dependent and maintained at all studied times. Endothelins also modified the kinetic and internalization of the NE neuronal transporter. In the anterior hypothalamic region endothelin-3 increased the V_max and the B_max whereas endothelin-1 decreased them. However, in the posterior hypothalamic region both endothelins diminished the V_max as well as B_max. Neither endothelin-1 nor endothelin-3 modified neuronal norepinephrine transporter K_d in the studied hypothalamic regions. These findings support that in the posterior hypothalamic region both endothelins diminished neuronal norepinephrine transporter activity by reducing the amine transporter expression on the plasmatic membrane. Conversely, in the anterior hypothalamic region endothelin-3 enhanced neuronal norepinephrine transporter activity by increasing the expression of the transporter on the presynaptic membrane, whereas endothelin-1 induced the opposite effect. Present results permit us to conclude that both endothelins play an important role in the regulation of norepinephrine neurotransmission at the presynaptic nerve endings in the hypothalamus.

Introduction

Endothelins (ETs) are peptides of 21 amino acids, that exist in three isoforms, ET-1, ET-2 and ET-3 and display vasoactive as well as growth regulatory properties (Kedzierski and Yanagisawa, 2001, Schneider et al., 2007). The biological actions of ETs are mediated by two well characterized G-protein coupled receptors, the ET_A receptor, that shows higher affinity for ET-1 than for ET-2 and ET-3, and the ET_B receptor, that displays similar affinity for the three isoforms (Motte et al., 2006, Schneider et al., 2007). However, several studies support the existence of atypical or non-conventional ET receptors based on atypical responses in the presence of selective ETs agonists and/or antagonists (Nambi et al., 1997, Henry and King, 1999, di Nunzio et al., 2004, Perfume et al., 2007, Perfume et al., 2008). Furthermore a third receptor subtype, termed ET_C was cloned in Xenopus laevis and although functional studies support its existence in mammals it has not been cloned in this specie yet (di Nunzio et al., 2004, Motte et al., 2006, Perfume et al., 2007, Schneider et al., 2007). The conventional ET receptors (ET_A and ET_B) by coupling to different G proteins, stimulate multiple intracellular signaling pathways including adenylyl cyclase/PKA, nitric oxide (NO)/guanylyl cyclase/PKG as well as the phosphoinositide pathway (Jaureguiberry et al., 2004, Motte et al., 2006, Schneider et al., 2007).

The ET system (precursor peptides, endothelin converting enzyme and receptors) is widely distributed in different mammalian tissues including the central nervous system (CNS) (Kuwaki et al., 1997, Schneider et al., 2007). In the hypothalamus the ET system is highly expressed suggesting that these peptides participate in the regulation of diverse biological functions controlled at this level (Kuwaki et al., 1997). The hypothalamus coordinates endocrine, neuroendocrine and autonomic signals involved in the control of the cardiovascular activity, water and sodium homeostasis as well as hormone release (Oparil et al., 1995, Hiller-Sturmhofel and Bartke, 1998). Among the different areas and nuclei that form the hypothalamus, the anterior and posterior hypothalamic regions play a major role in the regulation of the cardiovascular activity (Oparil et al., 1995). The anterior hypothalamic region (AHR) is a sympathoinhibitory area whereas the posterior region (PHR) behaves as a sympathoexcitatory area (Oparil et al., 1995). Functional impairment of these areas has been associated with hypertension (Oparil et al., 1995).

Since the work by Yanagisawa et al. (1988) who first reported the existence of ET, extensive studies have shown direct effects of ETs on the cardiovascular system, but substantially less works have been focused on the interaction between ETs and the brain. In this regard, it was shown that ET-1 increases dopamine release in the rat striatum whereas ET-3 augments catecholamine output in cortical as well as striatal brain slices (Koizumi et al., 1992; van den Buuse and Webber, 2000). We have previously reported that both ETs regulate neuronal NE release as well as the short-term and long-term modulation of tyrosine hydroxylase (TH) activity and expression of the phosphorylated forms of the enzyme in the AHR and PHR (di Nunzio et al., 2002, di Nunzio et al., 2004, Morgazo et al., 2005, Perfume et al., 2007, Perfume et al., 2008).

Neuronal norepinephrine (NE) uptake represents the primary means of inactivation of NE at noradrenergic synapses. The active transport of NE into presynaptic nerve endings after its neuronal release is mediated by the neuronal NE transporter (NET) (Blakely et al., 1994, Eisenhofer, 2001, Bönisch and Brüss, 2006). The NET belongs to a family of Na⁺/Cl⁻ dependent transporters which also includes transporters for serotonin, dopamine, glycine and GABA (Blakely et al., 1994, Eisenhofer, 2001, Bönisch and Brüss, 2006, Mandela and Ordway, 2006). NET is dependent on Na⁺ gradient, Cl⁻, ATP and temperature (Blakely et al., 1994, Eisenhofer, 2001, Bönisch and Brüss, 2006, Mandela and Ordway, 2006). NET activity may be acutely or chronically regulated by diverse stimuli including neuronal activity, neurotransmitters, hormones and second messengers elevated following receptor activation (Apparsundaram et al., 1998, Bryan-Lluka et al., 2001, Eisenhofer, 2001). In addition, impairment of NET has been reported in disorders such as hypertension and cardiomyopathy (Eisenhofer, 2001, Schlaich et al., 2004, Bönisch and Brüss, 2006, Mayer et al., 2006).

On these bases, in the present study we sought to establish if ET-1 and ET-3 also modulated NET activity in the hypothalamic regions related to the control of cardiovascular function such as the AHR and PHR. Our findings show that ET-1 in the AHR and PHR and ET-3 in the PHR reduced NET activation whereas ET-3 increased it in the AHR.