Pulmonary kinetic expression of the endothelin sys

Authors: A Zannoni C Bernardini F Gentilini M Giunti M L Bacci M Forni

Publish Date: 2010/05/02

Volume: 34, Issue: 1, Pages: 21-24

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Abstract

Endothelin ET1 is a potent vasoconstrictor peptide involved in the derangement of respiratory mechanics during endotoxic shock We measured the kinetics of pulmonary mRNA expression of the key components of the ET system ie ET1 ETconverting enzyme ECE and ETA and ETB receptors by quantitative realtime reverse transcriptasepolymerase chain reaction in a swine model of endotoxic shock 0 1 2 3 and 4 h of continuous LPS infusion at 40 μg/kg/hour sham group 4 hour saline infusion A significant increase in mRNA expression levels was observed for ET1 in LPStreated piglets the increase began as early as 1 hour In contrast no significant variations were observed for the ECE ETA or ETB genes Small gene expression differences observed with respect to our previous results suggest a possible effect of the anesthesia or surgical protocol on ET system regulationEndotoxic shock is a pathological condition associated with cardiac dysfunction pulmonary hypertension systemic hypotension and altered mechanical properties of the respiratory system Endothelin ET1 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 ET1 Dupuis et al 1996 ET1 is synthesized as a prepropeptide molecule and is converted into an active peptide by endothelin1 converting enzyme ECE1 ET1 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 ET1 ECE1 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 ET1 system in the lung an increase of ET1 mRNA and a decrease in the mRNA levels of the other components Since ET1 peptide has a short halflife 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 shockExperiments 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 130 ± 243 kg mean ± SE and about 2 months old were sedated with medetomidine hydrochloride Domitor 10 mL Pfizer 25 µg/Kg im tiletamine HCl and Zolazepam HCl Zoletil 100 Virbac 5 kg im and anesthetized with a bolus of 15 mg/kg thiopental sodium Pentothal Sodium Intervet 15 mg/kg iv 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 iv infusion of 40 μg/kg/h Escherichia coli LPS serotype 055B5 Sigma diluted in 09 sterile saline solution or saline solution only for 4 h sham group n = 2 Animals were then euthanized 6 mL Tanax® iv embutramide mebenzonium iodide and tetracaine hydrochloride and lung samples were taken for RNA extraction Realtime quantitative PCR was performed using an iCycler thermocycler BioRad Specific porcine primers for ET1 ECE1 ETA ETB and hypoxanthine phosphoribosyltransferase HPRT housekeeping gene were designed and realtime 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 oneway analysis of variance ANOVA p  005 was considered significantRelative gene expression of ET1 ECE1 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  005 ANOVA Duncan’s testThis study provides evidence that the surgical protocol utilized did not influence the gene expression levels of key components of the ET1 system in the lung LPS treatment was able to induce an increase in ET1 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 ET1 mRNA induction In contrast LPS did not influence the gene expression levels of ECE1 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

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