Cellular bioenergetics changes in magnocellular neurons may affect copeptin expression in the late phase of sepsis

https://doi.org/10.1016/j.jneuroim.2013.12.006Get rights and content

Highlights

  • Copeptin plays an important role in the correct structural formation of vasopressin.

  • Increased hypothalamic IL-1β and NO levels cause bioenergetics changes.

  • Apoptosis markers increased in magnocellular neurons during sepsis.

  • Decreased copeptin expression compromising AVP synthesis and secretion in the sepsis.

Abstract

We investigated whether inflammatory mediators during cecal ligation and puncture (CLP)-induced sepsis may diminish copeptin expression in magnocellular neurons, thus affecting arginine-vasopressin (AVP) synthesis. The transcript abundance of IL-1β, IL-1R1, iNOS and HIF-1α was continuously elevated. IL-1β, iNOS and cytochrome c protein levels progressively increased until 24 h. Immunostaining for these proteins was higher at 6 and 24 h, as also seen in the annexin-V assay, while copeptin was continuously decreased. This suggests that increased IL-1β and NO levels may cause significant bioenergetics changes in magnocellular neurons, affecting copeptin expression and compromising AVP synthesis and secretion in the late phase of sepsis.

Introduction

Clinical studies report that high arginine-vasopressin (AVP) plasma levels can be found in patients in the early phase of sepsis, in an attempt to restore blood pressure, which tends to decrease due to inflammatory mediators. Nonetheless, in the late phase, despite progressive hypotension, the plasma AVP levels are low, contributing to septic shock and death (Landry et al., 1997, Sharshar et al., 2003a). Moreover, infusion of a low dose of exogenous AVP decreases norepinephrine requirement in septic patients, while maintaining or increasing blood pressure, systemic resistance and urine output in vasodilatory shock (Holmes et al., 2001). There are also clinical studies indicating impaired baroreflex sensitivity (Holmes et al., 2001), depletion of neurohypophyseal hormone content (Holmes et al., 2001, Sharshar et al., 2002), overproduction of nitric oxide (NO) and oxidative stress in AVP neurons (Holmes et al., 2001) as reasons for the AVP secretion impairment. Corresponding findings were also seen in experimental sepsis in previous work from our group (Correa et al., 2007, Pancoto et al., 2008, Oliveira-Pelegrin et al., 2009, Oliveira-Pelegrin et al., 2013).

During sepsis, the excessive production and release of inflammatory mediators may affect AVP synthesis. By using cecal ligation and puncture (CLP) to induce sepsis, we in fact saw a decrease in AVP expression in the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus (Oliveira-Pelegrin et al., 2010a, Oliveira-Pelegrin et al., 2010b). Septic patients and rats were also reported to show changes in AVP content, as well as neuronal and glial apoptosis in regions related to autonomic control, including SON and PVN hypothalamic nuclei (Sharshar et al., 2003b, Sonneville et al., 2010). We recently reported an increased expression of cleaved caspase-3 in SON magnocellular neurons of CLP-induced septic rats (Oliveira-Pelegrin et al., 2013) suggesting that apoptosis was occurring in these neurons. Increased cytokine levels, particularly interleukin-1β (IL-1β), are thought to trigger the inducible isoform of NO synthase (iNOS) gene expression in the hypothalamus (Wong et al., 1996b, Wong et al., 1997). Once induced, iNOS produces large NO levels, which may act dually on mitochondrial bioenergetics affecting oxygen consumption and enhancing the generation of superoxide anions by decreasing the electron flow through cytochrome c oxidase. These changes may result in a “metabolic hypoxia” and hydrogen peroxide formation (Mander and Brown, 2004, Mander et al., 2005, Erusalimsky and Moncada, 2007), which may further stimulate iNOS expression and, consequently, an increase in NO levels (Guix et al., 2005). This metabolic hypoxia may also induce the expression and stability of the α subunit of hypoxia-induced factor 1 (HIF-1α) (Chavez et al., 2000, Sharp and Bernaudin, 2004, Erusalimsky and Moncada, 2007). Dimerization of HIF-1α with the constitutive HIF-1β subunit generates the functional transcription factor HIF-1, which regulates the expression of various genes involved in cellular energy metabolism and in the apoptosis pathway (Bruick, 2000, Sharp and Bernaudin, 2004).

Apoptosis can be triggered by various stimuli that activate the extrinsic and/or intrinsic pathway upstream of the caspase cascade. The extrinsic apoptosis pathway is induced by the activation of death receptors, which in turn belong to the tumor necrosis factor receptor superfamily (TNFRS). On the other hand, the intrinsic apoptosis pathway is mainly associated with mitochondrial and other intracellular stress signals (Sola et al., 2013). Oxidative stress promotes the movement of pro-apoptotic proteins to the mitochondrial surface, which changes the permeability of the mitochondrial membrane leading to transient pore formation and consequent release of proteins related to the activation of the intrinsic apoptosis pathway, such as cytochrome c (Mignotte and Vayssiere, 1998, Erusalimsky and Moncada, 2007). An early signal at this stage of the apoptosis process is the exposure of phosphatidylserine (PS), which can be detected by its affinity for annexin-V (van Engeland et al., 1998).

On the background of all this information we hypothesized that the cellular bioenergetics changes seen during sepsis could trigger alterations in synthesis of the AVP precursor, including that of copeptin, a C-terminal glycopeptide in the AVP precursor preprovasopressin. Copeptin plays an important role in the correct structural formation and proteolytic maturation of AVP (Barat et al., 2004, Struck et al., 2005, Morgenthaler et al., 2008). With this in mind, we analyzed the expression of oxidative stress and apoptosis markers in copeptin-AVP neurons of the SON and associated these with changes in AVP synthesis and basal plasma concentrations typically seen in the late phase of sepsis.

Section snippets

Animals

Male Wistar rats (250 ± 30 g) provided by the Animal Facility of the Campus of Ribeirão Preto, University of São Paulo, were housed in controlled temperature (25 ± 1 °C) and photoperiodic (12:12 h night: day cycle) conditions, with food (Nuvilab CR-1, NUVITAL) and tap water available ad libitum. All experimental protocols were approved and performed according to the guidelines of the Ethics Committee of the University of São Paulo—Campus Ribeirão Preto. Humane endpoints in shock research (Nemzek et

Results

Within few hours after surgery, all CLP animals developed the typical clinical signs of sepsis, such as, lethargy, piloerection and diarrhea. Sham animals remained active in their cages, as expected. The fold changes of transcripts for IL-1β, IL-1R1, iNOS and HIF-1α in magnocellular neurons of the SON following CLP-induced sepsis were higher than in sham animals, mainly so in the initial phase of sepsis (4 and 6 h) (Table 1). Protein levels assayed by Western blot analysis showed an increase for

Discussion

In the initial stage of AVP synthesis, a precursor protein of 164 amino acid residues is produced. This precursor protein is structurally composed of the signal peptide, the AVP peptide, neurophysin II (a carrier responsible for axonal transport of the hormone to the nucleus for neurohypophysis), and copeptin. The latter is a glycopeptide present in the C-terminal part of the AVP precursor. Copeptin plays an important role in the correct structural formation and proteolytic maturation of AVP,

Disclosure statement

The authors have nothing to disclose.

Acknowledgments

The authors thank, Aline de Souza Soares, Antonio Zanardo, Mariana Rossin Martinez and Nadir Martins Fernandes for the technical assistance. Sérgio Akira Uyemura provided the infrastructure for quantitative PCR. Financial support from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) is gratefully acknowledged.

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