Introduction

Endotoxic shock is a pathological condition associated with cardiac dysfunction, pulmonary hypertension, systemic hypotension, and altered mechanical properties of the respiratory system. Endothelin (ET)-1 is a potent vasoconstrictor peptide involved in the dysfunction of respiratory mechanics during endotoxic shock (Ohta et al. 1990). The peptide is primarily synthesized by a broad variety of cells, and the lung is the major site of both clearance and production of circulating ET-1 (Dupuis et al. 1996). ET-1 is synthesized as a prepropeptide molecule and is converted into an active peptide by endothelin-1 converting enzyme (ECE-1). ET-1 effects are mediated by at least two receptors, ETA and ETB, and their blockade abolishes pulmonary hypertension and worsens systemic hypotension induced by lipopolysaccharide (LPS) (Ciminaghi et al. 2003). The roles of the ET system’s key components (ET-1, ECE-1, ETA, and ETB) in sepsis are not yet fully clarified. Our previous study in a porcine model (Forni et al. 2005) showed that an LPS infusion (lasting 4 h) induced opposite effects on the various components of the ET-1 system in the lung: an increase of ET-1 mRNA and a decrease in the mRNA levels of the other components. Since ET-1 peptide has a short half-life (15–20 min) (Galiè et al. 2004), the aim of the present research was to evaluate lung ET system expression levels at different times after LPS treatment during porcine endotoxic shock.

Material and methods

Experiments were performed in accordance with local, national, and European laws for the care and use of laboratory animals. Twelve large, white piglets of both sexes, weighing 13.0 ± 2.43 kg (mean ± SE) and about 2 months old, were sedated with medetomidine hydrochloride (Domitor, 10 mL Pfizer, 25 µg/Kg i.m.), tiletamine HCl, and Zolazepam HCl (Zoletil 100, Virbac, 5 kg i.m.) and anesthetized with a bolus of 15 mg/kg thiopental sodium (Pentothal Sodium, Intervet, 15 mg/kg i.v.) injected into the auricular vein. Anesthesia was maintained by continuous infusion of thiopental sodium (9 mg/kg/h). The animals were orotracheally intubated and mechanically ventilated using a Siare Falcon 201/B ventilator with medical air (setting 15–20 breaths/min). Pigs were randomly assigned to receive 0, 1, 2, 3, and 4 h (T0, T1, T2, T3, and T4; n = 10) i.v. infusion of 40 μg/kg/h Escherichia coli LPS (serotype 055:B5, Sigma, diluted in 0.9% sterile saline solution) or saline solution only for 4 h (sham group, n = 2). Animals were then euthanized (6 mL Tanax® i.v., embutramide, mebenzonium iodide, and tetracaine hydrochloride), and lung samples were taken for RNA extraction. Real-time quantitative PCR was performed using an iCycler thermocycler (Bio-Rad). Specific porcine primers for ET-1, ECE-1, ETA, ETB, and hypoxanthine phosphoribosyltransferase (HPRT, housekeeping gene) were designed, and real-time PCR (SYBR green I) was performed as previously described (Forni et al. 2005). Relative gene expression levels were calculated using the 2−ΔΔCT method (Applied Biosystems User Bulletin #2, 2001) relative to the control T0. Statistical significances of differences were determined using the one-way analysis of variance (ANOVA; p < 0.05 was considered significant).

Results

The gene expression levels of ET-1, ECE-1, ETA, and ETB in the lung are shown in Fig. 1. Our results showed that LPS treatment induced a gradual increase, as early as 1 h, of ET-1 mRNA, reaching a maximum value after 4 h of LPS (12-fold increase with respect to control T0). ET-1 gene expression in the sham group did not change, showing that our surgical protocol did not influence gene expression levels. LPS treatment did not significantly influence ECE-1 gene expression; in the sham group, there was a slight increase in ECE-1 levels that was higher than with LPS treatment but this increase was not statistically significant. The gene expression levels of the receptors did not significantly change even with a slight increase in the levels of ETA and a slight decrease in the levels of ETB in both the treated and sham groups.

Fig. 1
figure 1

Relative gene expression of ET-1, ECE-1, ETA, and ETB in lungs of piglets after infusion with either LPS (1, 2, 3, and 4 h) or saline solution (sham 4 h). Gene expression levels were calculated as the fold increase relative to control (T0). Different letters indicate statistically significant differences (p < 0.05; ANOVA, Duncan’s test)

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Discussion

This study provides evidence that the surgical protocol utilized did not influence the gene expression levels of key components of the ET-1 system in the lung. LPS treatment was able to induce an increase in ET-1 gene expression in the lung. The increase began after 1 h of treatment and continued for 4 h, confirming that LPS is a strong stimulus for ET-1 mRNA induction. In contrast, LPS did not influence the gene expression levels of ECE-1, ETA, or ETB, in contrast to our previous results (Forni et al. 2005) in which LPS treatment at 4 h decreased their expression levels. These conflicting results suggest, together with the trends observed in the sham group, a possible effect of the anesthesiological/surgical protocol. Since assisted ventilation itself may influence pulmonary function to initiate or improve local and/or systemic inflammatory responses (Behnia et al. 1996; Uhlig 2002), it could also be responsible for the small differences in the expression levels of some ET system components observed during our studies.