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Published ahead of print on April 20, 2006, doi:10.1164/rccm.200508-1221OC
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American Journal of Respiratory and Critical Care Medicine Vol 174. pp. 198-207, (2006)
© 2006 American Thoracic Society
doi: 10.1164/rccm.200508-1221OC

Original Article

Dobutamine Improves Liver Function after Hemorrhagic Shock through Induction of Heme Oxygenase-1

Alexander Raddatz, Darius Kubulus, Johannes Winning, Inge Bauer, Sascha Pradarutti, Beate Wolf, Sascha Kreuer and Hauke Rensing

Department of Anesthesiology and Critical Care Medicine, University of the Saarland, Homburg, Germany

Correspondence and requests for reprints should be addressed to Hauke Rensing, M.D., Klinik für Anaesthesiologie und Intensivmedizin, Universität des Saarlandes, D-66421 Homburg/Saar, Germany. E-mail: aihren{at}uniklinik-saarland.de


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: Induction of heme oxygenase-1 (HO-1) protects the liver against reperfusion injury after hemorrhagic shock. Previous data suggest that the beta1-adrenoceptor agonist dobutamine induces HO-1 in hepatocytes.

Objectives: To investigate the functional significance of dobutamine pretreatment for liver function after hemorrhagic shock in vivo.

Methods: Anesthetized rats received either Ringer's (Vehicle/Shock), 10 µg/kg/min of the beta1-adrenoceptor agonist dobutamine (Dob/Shock), or 10 µg/kg/min dobutamine and 500 µg/kg/min of the beta1-adrenoceptor antagonist esmolol (Dob/Esmolol/Shock) for 6 h. Hemorrhagic shock was induced thereafter (mean arterial pressure, 35 mm Hg for 90 min). Animals were resuscitated with shed blood and Ringer's. In addition, the HO pathway was blocked after dobutamine pretreatment with 10 µmol/kg tin-mesoporphyrin-IX (Dob/SnMP/Shock) or animals received 100 mg/kg of the carbon monoxide donor dichloromethane (DCM/Shock).

Measurements: Hepatocellular metabolism and liver blood flow were measured by plasma disappearance rate of indocyanine green (PDRICG) as a sensitive marker of liver function.

Main Results: Pretreatment with dobutamine induced HO-1 in pericentral hepatocytes and improved PDRICG (Vehicle/Shock: 11.7 ± 8.12%/min vs. Dob/Shock: 19.7 ± 2.46%/min, p = 0.006). Blockade of the HO pathway after preconditioning and the combined pretreatment with dobutamine and esmolol decreased PDRICG (Dob/SnMP/Shock: 12.6 ± 4.24%/min, p = 0.011; Dob/Esmolol/Shock: 10.2 ± 4.34%/min, p = 0.008). Pretreatment with a carbon monoxide donor improved PDRICG (DCM/Shock: 18 ± 3.19%/min, p = 0.022) compared with Vehicle/Shock.

Conclusions: These results suggest a beta1-adrenoceptor–dependent hepatic up-regulation of HO-1 and a better maintained hepatocellular function after hemorrhagic shock in animals pretreated with dobutamine. The improved hepatocellular function may be in part mediated by carbon monoxide because of up-regulation of HO-1. Pretreatment with dobutamine might be a potential means of pharmacologic preconditioning before ischemia-reperfusion of the liver.

Key Words: dobutamine • heme oxygenase-1 • hemorrhagic shock • liver • preconditioning

Heme oxygenase (HO)-1 catalyzes the rate-limiting step in the degradation of heme to biliverdin, iron, and carbon monoxide (CO). Two different isoforms of HO have been characterized. Whereas the isoform HO-2 is constitutively expressed (1–3), the isoform HO-1 is highly inducible by a variety of stress conditions (4, 5).

Although biliverdin is a potent antioxidant, CO plays a pivotal role as a putative vasodilator for regulation of hepatic vascular resistance (6, 7), most notably under stressful conditions (8, 9). Hemorrhagic shock leads to a profound hepatocellular induction of HO-1 (5, 10). Blockade of the HO pathway after hemorrhagic shock increases hepatocellular injury (5), indicating a protective role of HO-1 under these conditions.

Further pathways of HO-1 induction under stress conditions are described. In addition to oxidative stress (5, 11), a protein kinase A–dependent induction of HO-1 was observed (12). Hepatocytes exhibit both {alpha}- and beta-adrenoceptors (13). Immunohistochemistry reveals a zonal expression of beta1-adrenoceptors in the pericentral region. Stimulation of beta1-adrenoceptors leads to a time- and dose-dependent induction of HO-1 primarily in pericentral hepatocytes (14).

The functional role of HO-1 induction after dobutamine treatment has not yet been clarified. The aim of this study is to assess the functional significance of dobutamine pretreatment for hepatic function and perfusion after hemorrhagic shock in vivo.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
All experiments were performed in accordance with the German legislation on protection of animals and the National Institutes of Health guidelines for animal care. Male Sprague-Dawley rats (200–250 g body weight) were obtained from Charles River (Sulzfeld, Germany).

Surgical Procedures
Animals were anesthetized and prepared as described previously (5). In addition, the right femoral vein was cannulated for continuous administration of indocyanine green (ICG). Cardiac output was measured by transpulmonary thermodilution technique (Cardiotherm 500; Columbus Instruments, Columbus, OH).

Hemorrhagic shock was induced by rapid arterial blood withdrawal by way of the left femoral artery (mean arterial pressure [MAP]: 35 ± 5 mm Hg for 90 min). Animals were resuscitated with 60% of the shed blood withdrawn infused during the first 10 min of resuscitation and twice the shed blood volume as Ringer's solution during the first hour of resuscitation. The infusion rate of Ringer's solution was lowered to a volume equaling the maximal bleed-out volume for the second hour of resuscitation. At the end of the experiment, livers were harvested and stored at –70°C until further processing.

Experimental Protocol
To study the dose-dependent induction of HO-1, animals (n = 6/group) were treated with 10, 20, or 50 µg/kg/min dobutamine for 6 h. At the end of the experiment, livers were harvested and stored at –70°C until further processing. In further control experiments, animals were treated with 10 µg/kg/min dobutamine for 6 h to assess the organ-specific expression of HO-1. Liver, heart, aorta, lung, kidney, and jejunum were harvested at the end of the experiment and stored at –70°C until further processing.

Sham-operated animals (n = 8) received a constant infusion of 10 ml/kg/h Ringer's solution during the entire period of the experiment but did not undergo hemorrhage.

Preconditioning Protocol
Animals received either 10 ml/kg/h Ringer's (Vehicle/Shock; n = 9) or 10 µg/kg/min dobutamine (Dob/Shock; n = 9) or a combination of 10 µg/kg/min dobutamine and 500 µg/kg/min of the beta1-adrenoceptor antagonist esmolol (Dob/Esmolol/Shock; n = 5) for 6 h before induction of hemorrhagic shock. Drug infusion was stopped 30 min before the onset of hemorrhagic shock.

In control experiments, the HO pathway was blocked after dobutamine pretreatment with the false substrate tin mesoporphyrin-IX (SnMP-IX; Porphyrin Products, Logan, UT) 15 min before induction of hemorrhagic shock (Dob/SnMP/Shock; n = 9). SnMP-IX was prepared and administered as described previously (15).

To assess the role of CO, animals received 100 mg/kg of the CO donor dichloromethane (DCM) 6 h before induction of shock (DCM/Shock; n = 9). DCM was administered by a stomach tube (16). CO blood content was measured spectrophotometrically at baseline and before induction of hemorrhagic shock.

Assessment of ICG Plasma Disappearance Rate
After 1 h of reperfusion, ICG (Pulsion, Munich, Germany) was continuously infused (5 mg/h) by the right femoral vein for 1 h to achieve a steady state. All syringes and tubes were covered with tinfoil to avoid phototoxicity. Animals were heparinized with 300 IE/kg body weight fifteen min before taking blood samples. Blood samples (0.3 ml) were taken at 0, 2, 4, 6, 8, 10, 15, and 20 min after switching off the perfusor and immediately centrifuged at 10,000 g for 5 min. ICG absorbances were determined spectrophotometrically at a wavelength of 800 nm. Measured ICG absorbances were converted into the corresponding plasma concentrations using a dose–response relationship. ICG plasma disappearance rate (PDRICG) was defined as the percentage decrease in ICG-plasma concentration per minute (%/min).

Quantitative Determination of Serum Enzyme Levels
Alanine aminotransferase (ALAT), glutamate dehydrogenase (GLDH), troponin T, lipase, and creatinine were analyzed with commercially available kits (Roche Diagnostics, Mannheim, Germany).

Western Blot Analysis and Immunohistochemistry
Western blot analysis and immunohistochemistry were performed as described previously (1).

Statistical Analysis
Data are presented as means ± SD. Differences were evaluated using analysis of variance followed by Student-Newman-Keuls test; p < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Dose-dependent Hepatocellular Induction of HO-1 after Dobutamine Treatment
In dose–response experiments, animals were treated with 10, 20, or 50 µg/kg/min dobutamine for 6 h or received 10 ml/kg/h Ringer's solution (sham). Untreated animals served as controls. HO-1 protein was barely detectable in livers from unmanipulated controls. A slight induction of HO-1 was observed in sham-operated animals, whereas a significant and dose-dependent increase of HO-1 immunoreactive protein after treatment with dobutamine was observed in vivo (Figure 1).


Figure 1
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  		Figure 1. Dose-dependent induction of heme oxygenase (HO)-1. Western blot analysis was performed as described. Aliquots of liver protein were fractionated by gel electrophoresis and electroblotted to polyvinylidene membranes. Membranes were incubated with an HO-1 primary antibody and the antigen antibody conjugate detection was achieved by an enhanced chemiluminescent reaction. Signal detection quantification was reached by a short exposure to film and subsequent densitometric analysis. Two protein samples of control animals, sham-operated animals and animals treated with different dosages of dobutamine for 6 h, are shown in a representative Western blot in A. The densitometric data are shown in B. HO-1 immunoreactive protein was below the detection limit in control animals. Only a slight HO-1 expression was observed in sham-operated animals. A dose-dependent increase in de novo HO-1 protein synthesis was observed after dobutamine treatment. Arbitrary densitometric units are given as mean ± SD. *p < 0.05 compared with sham.

 
Immunohistochemical Detection of HO-1 Expression
Cell-type–specific and spatial expression pattern of HO-1 on the protein level was studied by immunohistochemistry. Representative liver sections were obtained from time-matched sham-operated controls, animals treated with 10 µg/kg/min dobutamine, or animals treated with 50 µg/kg/min dobutamine for 6 h. Regarding the cell-type–specific and spatial expression pattern of HO-1, sham-operated control animals expressed only small amounts of HO-1 immunoreactive protein in nonparenchymal cells, primarily in periportal fields. HO-1 immunoreactive protein was barely detectable in hepatocytes of sham-operated control animals (Figures 2A and 2B). Treatment with dobutamine led to a dose-dependent and substantial de novo expression of HO-1 immunoreactive protein in parenchymal cells in the midzonal and pericentral region and tended to increase HO-1 immunoreactive protein in nonparenchymal cells (Figures 2C–2F).


Figure 2
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  		Figure 2. Cell type and spatial expression pattern of HO-1 in vehicle- and dobutamine-treated animals. Immunohistochemical detection of HO-1 immunoreactive protein was performed in paraffin-embedded dewaxed liver sections using a polyclonal HO-1 primary antibody. Representative liver sections were obtained from time-matched sham-operated control animals (A, B), animals treated with 10 µg/kg/min dobutamine (C, D), or animals treated with 50 µg/kg/min dobutamine (E, F) for 6 h. Regarding the cell-type–specific and spatial expression pattern of HO-1, sham-operated control animals expressed only small amounts of HO-1 immunoreactive protein in nonparenchymal cells, primarily in periportal fields. HO-1 immunoreactive protein was barely detectable in hepatocytes of sham-operated control animals (A, B). Treatment with dobutamine led to a dose-dependent and substantial de novo expression of HO-1 immunoreactive protein in parenchymal cells in the midzonal and pericentral region and tended to increase HO-1 immunoreactive protein in nonparenchymal cells (C–F). (A, C, E): original magnification, x10; (B, D, F): original magnification, x40. Asterisks mark central veins. Arrowheads mark periportal fields. White arrows mark hepatocytes. Black arrows mark nonparenchymal cells.

 
Organ-specific Induction of HO-1 after Dobutamine Treatment
In control experiments, animals were treated with 10 µg/kg/min dobutamine for 6 h. Compared with sham-operated control animals, a profound de novo synthesis of HO-1 immunoreactive protein was observed in liver, heart, aorta, lung, kidney, and jejunum after dobutamine treatment (Figure 3).


Figure 3
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  		Figure 3. Organ-specific induction of HO-1 after dobutamine pretreatment. Western blot analysis was performed as described previously. For each tissue, one representative sample after 6 h dobutamine pretreatment and one sample after 6 h vehicle treatment is shown in this Western blot. Spleen tissue served as positive control for HO-1 protein. The mean of the densitometric data of four animals are shown in the bar graphs. Dobutamine treatment led to a significant HO-1 induction in all investigated organs. Densitometric units are given as mean ± SD; *p < 0.05 compared with control.

 
Macrohemodynamic Parameters
Baseline values of MAP, heart rate, and cardiac output were comparable in all groups. Sham-operated animals exhibited normal and stable hemodynamic conditions throughout the experiment. Treatment with 10 µg/kg/min dobutamine led to a significant increase in heart rate (Figure 4A), whereas no significant differences in MAP were observed (Figure 4B). Cardiac output was increased during preconditioning with dobutamine (Figure 4C). A decrease in heart rate and cardiac output toward baseline was observed within 15 min after termination of dobutamine pretreatment. Dob/Esmolol/Shock animals showed comparable hemodynamic parameters with the Vehicle/Shock group. Before onset of hemorrhagic shock, all groups exhibited comparable macrohemodynamic conditions. Induction of hemorrhagic shock led to an initial profound decrease in heart rate in Vehicle/Shock animals, whereas it decreased only slightly in the Dob/Shock group (Figure 4A). Hemorrhagic shock was reversible in all animals as reflected by recovery of MAP, heart rate, and cardiac output after retransfusion of shed blood. Shed blood volumes (milliliter per kilogram) were comparable in all groups subjected to hemorrhagic shock (Table 1), suggesting a comparable insult. During reperfusion, macrohemodynamic parameters were stable in all groups (Figure 4).


Figure 4
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  		Figure 4. Hemodynamic effects of dobutamine preconditioning. Heart rate (A), mean arterial pressure (MAP) (B), and relative changes in cardiac output (C). Dobutamine pretreatment led to a significant increase in heart rate and cardiac output, whereas no significant changes in MAP were observed. Vehicle-treated animals and Dob/Esmolol/Shock animals exhibited stable hemodynamic parameters during preconditioning. After termination of dobutamine administration, heart rate and cardiac output in the Dob/Shock group returned to baseline. At the onset of hemorrhagic shock, all groups had comparable hemodynamics. Induction of hemorrhagic shock led to a significant decrease in MAP and cardiac output in all groups. A significant decrease in heart rate was observed in Vehicle/Shock animals, whereas it remained stable in dobutamine-pretreated animals. After retransfusion of shed blood heart rate, MAP and cardiac output returned to baseline values in all groups. No significant differences in MAP between the groups were observed during the entire experiment. Cardiac output was calculated as relative changes of baseline values. Data are shown as mean ± SD of nine (Vehicle/Shock, Dob/Shock) or five (Dob/Esmolol/Shock) independent experiments. #p < 0.05 compared with Vehicle/Shock; $p < 0.05 compared with baseline.

 

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  		TABLE 1. SHED BLOOD VOLUME

 
Blockade of the HO pathway with SnMP in dobutamine-treated animals (Dob/SnMP/Shock) had no significant influence on macrohemodynamic parameters like MAP, heart rate, and cardiac output compared with dobutamine-treated animals without blockade (data not shown). Administration of DCM (DCM/Shock) did not lead to significant changes in MAP, heart rate, and cardiac output compared with the Vehicle/Shock group (data not shown).

Blood Gas Analysis
Blood gas analysis was performed immediately after blood sampling. Baseline values of respiratory parameters (PO2, PCO2), acid-base metabolism (pH, base excess), and hemoglobin content were comparable in all groups. A significant reduction of base excess was observed in all shock groups indicating a comparable severity of hemorrhagic shock. Base excess recovered during resuscitation in all shock groups (Table 2).


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  		TABLE 2. BLOOD GAS ANALYSIS

 
PDRICG
Hemorrhagic shock and resuscitation significantly impaired PDRICG compared with sham-operated animals. Pretreatment with dobutamine before induction of hemorrhagic shock improved PDRICG. A combined pretreatment with dobutamine and esmolol impaired PDRICG after hemorrhagic shock compared with dobutamine alone. Blockade of the HO pathway with SnMP-IX after dobutamine treatment abolished the observed protection. Application of DCM before shock led to a significant increase in CO hemoglobin (from 3.5 ± 0.56% to 15.1 ± 3.51%, p < 0.001) and improved PDRICG (Figure 5).


Figure 5
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  		Figure 5. Plasma disappearance rate of indocyanine green (PDRICG) after 2 h of resuscitation from hemorrhagic shock. ICG absorbances were determined spectrophotometrically at a wavelength of 800 nm in plasma samples. Measured ICG absorbances were converted into the corresponding plasma concentrations using a dose–response relationship. PDRICG was defined as the percentage decrease in ICG plasma concentration per minute (%/min). Hemorrhagic shock (Vehicle/Shock; n = 9) led to a significant impairment of PDRICG compared with sham-operated animals. Pretreatment with the beta1-adrenoceptor agonist dobutamine (Dob/Shock; n = 9) before hemorrhagic shock significantly improved PDRICG after reperfusion. Blockade of beta1-adrenoceptors with esmolol (Dob/Esmolol/Shock; n = 5) during dobutamine-pretreatment or blockade of the HO-pathway with SnMP-IX (Dob/SnMP/Shock; n = 9) prevented the observed protection through dobutamine. Pretreatment with the carbon monoxide donor dichloromethane (DCM; DCM/Shock; n = 9) significantly increased PDRICG compared with Vehicle/Shock animals. Data are shown as mean ± SD. *p < 0.05 compared with sham; #p < 0.05 compared with Vehicle/Shock; §p < 0.05 compared with Dob/Shock.

 
beta1-Adrenoceptor–dependent Induction of HO-1
Samples of sham-operated, vehicle-treated, and dobutamine-treated (10 µg/kg/min) animals and animals treated simultaneously with dobutamine (10 µg/kg/min) and esmolol (500 µg/kg/min) were analyzed. Dobutamine treatment for 6 h led to a significant increase of HO-1 expression compared with sham-operated animals. Blockade of beta1-adrenoceptors with the beta1-adrenoceptor antagonist esmolol prevented this increase (Figure 6).


Figure 6
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  		Figure 6. beta1-Adrenoceptor–dependent induction of HO-1. (A) A representative Western blot. Liver protein samples of sham-, vehicle-, dobutamine- (10 µg/kg/min), or dobutamine- (10 µg/kg/min) and esmolol- (500 µg/kg/min) treated animals were used. (B) The densitometric data. Dobutamine treatment led to a significant increase in HO-1 expression compared with sham-operated animals. This increase was prevented by simultaneous blockade of beta1-adrenoceptors during dobutamine-treatment. Data are presented as mean ± SD. *p < 0.05 compared with sham.

 
Influence of DCM Pretreatment on HO-1 Induction
In further control experiments, the influence of DCM pretreatment on HO-1 induction in the liver was investigated to assess possible effects of the reagent on hemoglobin inhibiting oxygen delivery and enhancing hypoxic preconditioning. No significant induction of HO-1 was observed after DCM treatment, making a hypoxic preconditioning through DCM pretreatment unlikely (Figure 7).


Figure 7
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  		Figure 7. Influence of DCM on hepatocellular HO-1 expression. (A) A representative Western blot. Two representative liver protein samples of sham-, vehicle-, and DCM-treated animals were used. (B) The densitometric data. No significant differences in HO-1 induction were observed between sham-, vehicle-, and DCM-treated animals after 6 h.

 
Determination of Serum Enzyme Levels as Markers of Injury
Hepatocellular injury was assessed by measurement of serum enzyme levels of ALAT and GLDH.

Troponin T was measured as a marker of cardiac injury. Lipase serum enzyme levels were assessed as a marker of pancreatic injury. Creatinine levels were used as a marker of renal injury. Hemorrhage and subsequent resuscitation led to a significant increase in ALAT and GLDH activity in Vehicle/Shock and Dob/Shock animals. No significant differences were observed between both groups (Figure 8).


Figure 8
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  		Figure 8. Hepatocellular injury was assessed by plasma concentrations of alanine aminotransferase (ALAT; A) and glutamate dehydrogenase (GLDH; B). Pretreatment with dobutamine did not increase plasma levels of ALAT or GLDH. Hemorrhagic shock and reperfusion led to a significant increase in GLDH and ALAT activity. No significant differences between vehicle- and dobutamine-treated animals were observed. Data are shown as mean ± SD. $p < 0.05 compared with baseline.

 
Pretreatment with dobutamine decreased serum enzyme levels of troponin T compared with vehicle, indicating less cardiac injury after dobutamine pretreatment (Figure 9). No significant changes were observed for creatinine or lipase serum enzyme levels.


Figure 9
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  		Figure 9. Cardiac injury was estimated by measurement of troponin serum enzyme levels (A), pancreatic injury was estimated by serum enzyme levels of lipase (B), and renal injury was estimated by measurement of serum enzyme levels of creatinine (C). Serum levels of troponin at the end of the experiment were lower in dobutamine-treated animals compared with the vehicle group (A). There was no difference in serum creatinine and lipase levels between dobutamine- and vehicle-treated animals at the end of the experiment (C). $p < 0.05 compared with baseline; #p < 0.05 compared with vehicle.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study investigated the functional significance of pretreatment with the beta1-adrenoceptor agonist dobutamine before hemorrhage and resuscitation on liver function and perfusion. Preconditioning with dobutamine led to a hepatocellular HO-1 induction and significantly increased PDRICG after hemorrhagic shock. This protective effect was abrogated by blocking the HO pathway with SnMP-IX. Blockade of beta1-adrenoceptors with esmolol during dobutamine preconditioning attenuated induction of HO-1 and PDRICG. Administration of a CO donor before induction of hemorrhagic shock improved PDRICG. These results suggest a beta1-adrenoceptor–dependent up-regulation of HO-1 in the liver through pretreatment with dobutamine. This up-regulation of HO-1 after preconditioning contributes to an improved hepatic function and perfusion after hemorrhagic shock, which might be in part mediated through the increased HO-dependent production of CO. Furthermore, induction of HO-1 is observed in several other organs, such as heart, aorta, lung, jejunum, and kidneys, after 6 h of dobutamine pretreatment. Looking at markers of injury, a decrease of troponin serum enzyme levels after dobutamine pretreatment indicates a protective role for cardiac injury after shock.

The phenomenon that pre-exposure of the liver to transient sublethal stress increases the tolerance to reperfusion injury is known as "hepatic preconditioning." Organ preconditioning is an emerging means of protecting the body against ischemia-induced injury as a consequence of surgical procedures. Several preconditioning protocols are reported, including brief ischemia followed by reperfusion (17, 18), whole-body hyperthermia (19–21), and chemical induction of heat shock proteins with geranylgeranylacetone (22–24) and cobalt-protoporphyrin (25). Hepatic preinduction of HO-1 with cobalt-protoporphyrin or with adenoviral HO-1 gene transfer before ischemia-reperfusion improved hepatocellular function in vivo (26).

Substantial disadvantages of the described ways of preconditioning include serious side effects (protoporphyrins) or technically complex protocols (adenoviral HO-1 gene transfer). Pretreatment with dobutamine offers the opportunity of a receptor-dependent pharmacologic preconditioning before ischemia-reperfusion. Dobutamine is a preferential beta1-agonist commonly used for cardiac support in the case of myocardial dysfunction. Recent evidence suggests that treatment of brain-dead donors with catecholamines reduces the rejection risk and improves long-term transplantation outcome (27, 28). The mechanisms are not fully understood but may include up-regulation of HO-1. In several transplant models, preinduction of HO-1 by metalloporphyrins before cold ischemia has been shown to limit injury in the post-transplant period (29). In the present study, pretreatment with dobutamine was well tolerated and did not increase parameters of liver injury. The observed hemodynamic changes include tachycardia and an increase in cardiac output. These changes were reversible after stopping the dobutamine infusion. Dobutamine pretreatment dose-dependently induced HO-1 in pericentral hepatocytes. The observed zonal expression pattern of HO-1 correlates with the previously described zonal expression pattern of beta1-adrenoceptors in the liver (30). To avoid the possibility that dobutamine might directly influence hepatocellular function and sinusoidal perfusion by raising cardiac output and liver blood flow, administration of the drug was terminated 30 min before the onset of hemorrhagic shock. Considering the short half-life of dobutamine (31), a significant influence on systemic hemodynamics is unlikely. Furthermore, cardiac output at the beginning and at the end of shock was comparable between dobutamine-pretreated animals and those without pretreatment, indicating a comparable macrohemodynamic situation. A direct adrenergic effect of dobutamine on hepatic function and perfusion reflected by PDRICG is unlikely.

The question whether a post-shock treatment with dobutamine may lead to protection, as observed with the pretreatment, cannot be answered with the current data. Hemorrhagic shock and resuscitation lead to a profound induction of HO-1. Induction of HO-1 under these conditions contributes to maintenance of liver blood flow and hepatocellular integrity. The time course of HO-1 induction reaches a maximum 6 h after shock (32). Treatment with dobutamine after shock might further increase the shock-dependent induction of HO-1 and might possibly increase the protection already observed. An essential experimental problem to assess the protective effect of post-treatment is the increase in cardiac output through dobutamine. To differentiate between the effects of dobutamine on HO-1 induction and the positive effects of dobutamine on hemodynamics might be quite difficult under these conditions.

The organ-specific expression of HO-1 after dobutamine pretreatment showed an induction in the liver, heart, aorta, lung, kidney, and jejunum compared with sham-operated control animals. Serum enzyme levels after hemorrhagic shock with or without dobutamine pretreatment, including the serum enzyme levels of troponin T as a marker of cardiac injury, the enzyme levels of lipase as a marker of pancreatic injury, and the serum enzyme levels of creatinine as a marker of kidney function, showed unchanged creatinine and lipase levels, whereas troponin T was lower after dobutamine pretreatment. These data indicate a protective role of dobutamine pretreatment for cardiac injury after shock apart from the protective effects observed on liver function.

Different pathways of HO-1 induction under stress conditions have been described. Generation of reactive oxygen species is one possible mechanism to induce HO-1 (32). Immenschuh and coworkers (12) described a protein kinase A–dependent induction of HO-1 in vitro. In addition, other receptor-dependent pathways affect intracellular cAMP levels. In a previous study, the authors found a cAMP-dependent hepatocellular HO-1 induction after application of the selective beta1-adrenoceptor agonists dobutamine and xamoterol in vitro and in vivo (14). These data are in line with the observed induction of HO-1 after dobutamine pretreatment in the present study. Dobutamine pretreatment before onset of hemorrhagic shock increased PDRICG after hemorrhage, indicating a protective effect.

To assess the role of beta1-adrenoceptors for HO-1 induction, beta1-adrenoceptors were blocked with the antagonist esmolol during dobutamine preconditioning. Esmolol attenuated induction of HO-1 through dobutamine and decreased PDRICG after hemorrhagic shock. Esmolol is a beta1-selective adrenergic receptor blocking agent with a very short duration of action, and no significant intrinsic sympathomimetic activity at therapeutic dosages. It is rapidly metabolized by hydrolysis of the ester linkage by the esterases in the cytosol of red blood cells; the metabolism of esmolol is not limited by the rate of blood flow to metabolizing tissues. At the onset of hemorrhagic shock, 30 min after stopping the infusion of esmolol and dobutamine, a significant activity of these agents at beta1-adrenoceptors is unlikely. The observed attenuated HO-1 induction after preconditioning with dobutamine and esmolol and the decreased PDRICG after hemorrhagic shock under these conditions are likely to be dependent on an attenuated beta1-adrenoceptor activation in the presence of esmolol compared with dobutamine alone. Pretreatment with dobutamine is likely to induce HO-1 by a beta1-adrenoceptor–dependent mechanism and seems to be protective regarding liver function after hemorrhagic shock.

Microcirculatory disturbances as consequences of ischemia-reperfusion have a serious impact on reperfusion injury (33, 34). Although the underlying mechanisms are not completely understood, recent evidence suggests a dysregulation of vasoactive mediators during resuscitation. A complex interaction of endothelins, catecholamines, and gaseous oxides regulates liver blood flow under these conditions. The imbalance between vasodilators and vasoconstrictors has been identified as a potential target for therapeutic interventions (35). HO-1 is induced after hemorrhage and resuscitation (5, 10) and plays an essential role in protecting the liver against ischemia-reperfusion injury (5, 8). Mechanisms contributing to the observed protection through HO-1 may include the vasodilatory potential of CO (9, 10). Under stress conditions, HO-1 becomes the major site of CO generation (36). Consistent with this concept, the use of SnMP-IX as a specific blocker of the HO-CO pathway impaired portal (10) and sinusoidal blood flow (8) and led to an increased hepatocellular injury (5).

In the present study, the authors observed an induction of HO-1 after pretreatment with a beta1-adrenoceptor agonist accompanied by an improved liver function and perfusion after shock. Blockade of the HO pathway under these conditions abolished the observed protection after pretreatment. In control experiments animals were fed with the CO donor DCM to estimate the role of CO as a mediator of protection. DCM increased significantly CO hemoglobin levels and improved PDRICG compared with vehicle. Up-regulation of HO-1 after stimulation of beta1-adrenoceptors contributes to an improved hepatic perfusion and function after hemorrhagic shock, which might be mediated in part through the increased HO-dependent production of CO.

A decrease in ALAT and GLDH serum levels after preconditioning could not be observed but might be explained by an improved sinusoidal blood flow and subsequent flush out of liver enzymes into the systemic circulation in dobutamine-treated animals. In low-flow situations like hemorrhagic shock, PDRICG reflects hepatocellular function and liver perfusion more reliably than serum liver enzyme levels (37, 38). Systemic hemodynamics provides a poor estimate of hepatic perfusion. Measurement of PDRICG delivers information about hepatic perfusion and metabolism (39, 40). ICG is a nontoxic dye, which is eliminated exclusively by the liver in unaltered form without enterohepatic circulation (41, 42). PDRICG is a well-established quantitative marker of liver function in intensive care units. After intravenous administration, ICG is immediately bound to plasma proteins and is confined to the vascular compartment. PDRICG is directly influenced by hepatic blood flow and hepatocellular metabolism. In case of a reduced metabolic capacity or a dose-dependent saturation of hepatic dye elimination capability, the flow-dependency of ICG elimination is no longer crucial and PDRICG becomes dependent on hepatocellular metabolism. An improvement in gastric mucosal perfusion after dobutamine administration in patients with septic shock is reported in several studies (43–47). This improvement might be the result of a receptor-mediated redistribution of blood flow from the muscularis to the mucosa (48). Data suggest that dobutamine by itself has no relevant effect on hepatocytic clearance of ICG (48, 49).

In summary, the results indicate a beta1-adrenoceptor–dependent induction of HO-1, resulting in a protection of hepatic function and perfusion during hemorrhagic shock. In line with these results, dobutamine pretreatment might be a potential means to induce HO-1 in expected settings of ischemia-reperfusion events after major liver surgery or transplantation. Furthermore, the ability of beta1-agonists to stimulate the HO-catalyzed production of the vasodilator CO may play a role in liver blood flow regulation (e.g., during dobutamine therapy in critically ill patients). beta1-Agonists may be a useful pharmacologic tool to modulate hepatocellular induction of HO-1 in vivo to provide pharmacologic preconditioning.


   FOOTNOTES
 
Originally Published in Press as DOI: 10.1164/rccm.200508-1221OC on April 20, 2006

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form August 8, 2005; accepted in final form April 13, 2006


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