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SHOCK, Vol. 27, No. 6, pp. 687Y694, 2007
CYCLIC ADENOSINE MONOPHOSPHATEYPHOSPHODIESTERASE
INHIBITORS REDUCE SKELETAL MUSCLE PROTEIN
CATABOLISM IN SEPTIC RATS
Eduardo Carvalho Lira,* Flávia Aparecida Grac$ a,*
Dawit Albieiro P. Gonc$ alves,* Neusa M. Zanon,† Amanda Martins Baviera,†
Lena Strindberg,‡ Peter Lönnroth,† Renato Hélios Migliorini,† Isis C. Kettelhut,†
and Luiz Carlos C. Navegantes*†
*Department of Molecular Biology, São José do Rio Preto Medical School; † Departments of Physiology,
Biochemistry and Immunology, Ribeirão Preto Medical School, University of São Paulo, Brazil;
and ‡ Lundberg Laboratory for Diabetes Research, Department of Internal Medicine,
Sahlgrenska University Hospital, Göteborg, Sweden
Received 12 Jul 2006; first review completed 2 Aug 2006; accepted in final form 12 Oct 2006
ABSTRACT—We have previously shown that catecholamines exert an inhibitory effect on muscle protein degradation
through a pathway involving the cyclic adenosine monophosphate (cAMP) cascade in normal rats. In the present work, we
investigated in vivo and in vitro effects of cAMP-phosphodiesterase inhibitors on protein metabolism in skeletal muscle
from rats submitted to a model of acute sepsis. The in vivo muscle protein metabolism was evaluated indirectly by
measurements of the tyrosine interstitial concentration using microdialysis. Muscle blood flow (MBF) was monitored by
ethanol perfusion technique. Sepsis was induced by cecal ligation and puncture and resulted in lactate acidosis,
hypotension, and reduction in MBF (j30%; P G 0.05). Three-hour septic rats showed an increase in muscle interstitial
tyrosine concentration (~150%), in arterial plasma tyrosine levels (~50%), and in interstitial-arterial tyrosine concentration
difference (~200%; P G 0.05). Pentoxifylline (50 mg/kg of body weight, i.v.) infusion during 1 h after cecal ligation and
puncture prevented the tumor necrosis factor ! increase and significantly reduced by 50% (P G 0.05) the interstitial-arterial
tyrosine difference concentration. In situ perfusion with isobutylmethylxanthine (IBMX; 10j3 M) reduced by 40% (P G 0.05)
the muscle interstitial tyrosine in both sham-operated and septic rats. Neither pentoxifylline nor IBMX altered MBF. The
addition of IBMX (10j3 M) to the incubation medium increased (P G 0.05) muscle cAMP levels and reduced proteolysis in
both groups. The in vitro addition of H89, a protein kinase A inhibitor, completely blocked the antiproteolytic effect of IBMX.
The data show that activation of cAMP-dependent pathways and protein kinase A reduces muscle protein catabolism
during basal and septic state.
KEYWORDS—Microdialysis, sepsis, skeletal muscle, proteolysis, cAMP
INTRODUCTION
way (4, 5). The reduction in the activity of this proteolytic
system induced by catecholamines, clenbuterol (selective "2adrenergic agonist), CL 316,243 (selective "3-adrenergic agonist), and dibutyryl cyclic adenosine monophosphate (cAMP) in
vitro suggests that catecholamines inhibit proteolysis by binding
to "2 and "3-adrenoceptors and activating intracellular pathways
involving the cAMP cascade (4Y6). The understanding of the
precise mechanisms by which activation of cAMP-mediated
pathways promotes muscle anabolic effects may bring new
perspectives for efficient treatment of muscle-wasting conditions.
Drugs that induce an increase in the intracellular concentrations
of cAMP, as the nonspecific cAMP-phosphodiesterase inhibitors
(e.g., pentoxifylline), have been used in the treatment of various
peripheral vascular disorders characterized by an inadequate
tissue perfusion (7, 8). More recently, attention has focused on
the therapeutic potential of these drugs in preventing muscular
atrophy in several experimental situations, including nerve
damage (9), cancer (10), and sepsis (11, 12). A large number
of these studies have been performed in young rats because they
possess muscles that are thin enough to allow an adequate
diffusion of oxygen and substrates under in vitro conditions (13).
Although these approaches have unequivocally provided the
basis of our contemporary understanding of the regulation of
protein turnover in muscle, they often fail to accurately reflect
the in vivo condition. Microdialysis has raised high expectations
that it can meet the demand for a method that allows mechanistic
The loss of skeletal muscle protein is often a consequence
of diseases such as cancer, renal failure, AIDS, and sepsis.
Despite recent better understanding of the metabolic and
molecular derangements leading to muscle wasting, currently,
there are no safe and effective medicines available to treat
muscle atrophy. Loss of muscle mass results from increased
protein breakdown, decreased protein synthesis, or simultaneous changes in both processes. These pathways are
regulated by a set of signaling molecules, including hormones,
cytokines, nutrients, and neurotransmitters (1).
Among the factors that regulate skeletal muscle protein
metabolism, the sympathetic nervous system has an important
physiological role (2). In previous studies, we have found that
guanethidine-induced adrenergic blockade increases the rate of
protein degradation and decreases protein synthesis in rat soleus
muscles after 2 days of treatment, suggesting that catecholamines
exert an anabolic effect on oxidative muscle protein metabolism
(3). The activation of overall proteolysis was accompanied by an
increased participation of the Ca2+-dependent proteolytic pathAddress reprint requests to Luiz Carlos Carvalho Navegantes, Department
of Physiology, School of Medicine, USP, 14049-900 Ribeirão Preto, SP, Brazil.
E-mail: [email protected].
DOI: 10.1097/SHK.0b013e31802e43a6
Copyright Ó 2007 by the Shock Society
687
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SHOCK VOL. 27, NO. 6
investigations to be performed in human and animal skeletal
muscle (14). Thus, direct measurements of amino acid concentrations in muscle interstitial space by microdialysis can provide
important additional information on physiological and pathological processes in muscle tissues in vivo.
The purpose of the present work was to use microdialysis to
obtain information about the effect of cAMP-phosphodiesterase
inhibitors (isobutylmethylxanthine and pentoxifylline) on the
release of tyrosine from rat skeletal muscle in a classical
situation of muscle protein catabolism, cecal ligation and
puncture (CLP). Interstitial tyrosine concentration was measured
in tibialis anterior muscles of acutely septic rats systemically
treated with pentoxifylline (PTX) or perfused in situ with
isobutylmethylxanthine (IBMX). To determine the vasoactive
effects of these compounds, the measurements of muscle blood
flow (MBF) were performed with the microdialysis ethanol
technique (15). The direct in vitro effect of IBMX on the rate of
overall proteolysis was investigated in the presence or absence
of H89, an inhibitor of protein kinase A (PKA). The
concentration of cAMP in muscles from IBMX-treated rats in
vitro is also reported.
MATERIALS AND METHODS
Animals and experimental model—This study was approved by the Ethical
Committee on Animal Research of São José do Rio Preto Medical School (CEEA;
Proc. no. 6040/03). Male Wistar rats (~250 g) were used in all microdialysis
experiments. Because the incubation procedure required intact muscles sufficiently
thin to allow an adequate diffusion of metabolites and oxygen, young rats (~70 g)
were used in in vitro experiments. Rats were housed in a room with a 12-h/12-h
light/dark cycle and were given free access to water and normal laboratory chow
diet for at least 1 day before the beginning of the experiments, which were
performed at 8 AM.
Sepsis was induced by CLP as previously described (16). In brief, rats were
anesthetized with sodium thionembutal (50 mg/kg of body wt, i.p.). An abdominal
incision was made approximately 2 cm long, sufficient to expose the cecum and
adjoining intestine. With 4.0 silk suture, the cecum was tightly ligated just below
the ileocecal valve without causing bowel obstruction. The cecum was punctured
twice with 16-gauge needle near to ligature and was softly squeezed to extrude
feces and to ensure that the 2 puncture holes did not close. The incision was closed
with 4.0 nylon suture. At the end of surgery, 10 mL saline/100 g of body weight
was injected subcutaneously on the back to prevent hypovolemia and septic shock.
Control rats underwent sham operation, that is, laparotomy and manipulation, but
no ligation or puncture of the cecum.
A carotid artery catheter (PE-50; Becton Dickinson, USA) was placed through
an anterior cervical incision in the posterior cervical area. Unless otherwise
indicated, arterial blood samples were collected 3 h after CLP to measure pH, PO2,
PCO2, and HCO3j using a blood gas analyzer (ABL 505, EML 100; Radiometer,
Copenhagen NV, Denmark). Glucose and lactate concentrations were determined
using a YSI 2700 select biochemical analyzer (Yellow Springs, Ohio). Muscle and
liver glycogen content was measured by the method of Carrol et al. (17) in a
separate group of sham-operated and CLP animals.
Microdialysis studies
General procedure—One hour after CLP or sham operation, rats were placed
on heating pads to maintain temperature at 37-C. The trachea was cannulated to
facilitate respiration. A polyethylene catheter (PE-50; Becton Dickinson) was
placed in the left carotid artery to permit withdrawal of blood samples and
measurement of blood pressure. A microdialysis probe was inserted in the tibialis
anterior muscle, and an equilibration period of 30 min was allowed. The principle
of microdialysis has earlier been described in detail (18). In the present study,
catheters of single dialysis tubing (18 0.3 mm, Gambro, Cuprophane,
3000Ymolecular weight cutoff, Sweden) were glued to polyethylene tubing
(standardized length of 50 mm). After connecting the catheter inlet to a
microinjection pump (Insight 2000, Brazil), the system was perfused with 0.5%
bovine albumin, 1 mmol/L glucose, and 50 mmol/L tyrosine in isotonic saline at a
rate of 1.0 2L/min.
To investigate if all parameters analyzed (recovery, interstitial, and arterial
tyrosine) were stabilized along the time after insertion of the microdialysis
LIRA
ET AL.
catheter, a preliminary study was performed in normal control rats that were
followed for 240 min, with collection of perfusate (50 2L), muscle dialysate (50
2L), and arterial blood (300 2L) samples taken at each 80 min of experiment. In
the following experiments, samples for tyrosine measurements were collected at
the end of 90 min of microdialysis. Tyrosine was assayed by a fluorometric method
(19). Because skeletal muscle cannot synthesize or degrade tyrosine, its concentration in muscle reflects the balance between protein synthesis and degradation
(14). An increase in the concentration of tyrosine in the interstitium would
therefore indicate a shift in the balance toward net degradation.
In vivo probe recovery of tyrosine was assessed according to the internal
reference calibration technique and was determined in all samples (20).
Experimental protocol
cAMP-phosphodiesterase inhibitors and microdialysis studies—To investigate
the systemic effect of PTX (Sigma, St Louis, Mo), a catheter was inserted into the
left jugular vein for administration of the drug. Pentoxifylline (50 mg/kg of body
weight) was infused at the rate of 17 2L/min during 1 h immediately after CLP or
sham operation. Pentoxifylline dose was decided on the basis of previous studies
from other laboratories showing that the long-term treatment with this compound
reduces protein catabolism during sepsis and in other models of muscle atrophy
(10, 11). Control animals received a isovolumetric injection of vehicle (saline).
Three hours after CLP or sham surgery, blood was collected from abdominal aorta,
centrifuged (500g for 10 min at 4-C), and supernatants were used for determination
of tumor necrosis factor ! (TNF-!) levels.
To study the in situ effects of the phosphodiesterase inhibitors, muscles were
perfused during 90 min with IBMX (Sigma) or PTX (10j3 M) dissolved in the
same perfusion medium described above. This concentration has been shown to
induce a maximum antiproteolytic effect on skeletal muscle from normal rats in
vitro (5). Contralateral muscles were used as controls and were perfused with the
perfusion medium without IBMX.
IBMX and muscle proteolysis studies—To investigate the in vitro effect of
IBMX on the rate of overall proteolysis, skeletal muscles from sham-operated and
3-h septic rats were incubated in the presence of different concentrations of this
drug (10j5, 10j4, and 10j3 M) using the procedure described below. In a
separated group of experiments, the rate of overall proteolysis was also
investigated in muscles incubated in the presence of 10j3 M IBMX and
50 2mol/L H89 (N-(2-[p-bromocinnamylamino]-ethyl)-5-dulfonomide. 2 HCl,
Alexis Biochemicals), a specific PKA inhibitor.
Incubation procedure—Rats were killed by cervical dislocation for muscle
excision. The extensor digitorum longus (EDL) was rapidly dissected, care being
taken to avoid damaging the muscle. Extensor digitorum longus was maintained at
approximately resting length by pinning them on inert plastic supports. Tissues
were incubated at 37-C in Krebs-Ringer bicarbonate buffer, pH 7.4, equilibrated
with 95% oxygen 5% carbon dioxide, containing glucose (5 mmol/L) in the
absence or presence of IBMX (10j3 M).
Measurement of rates of protein degradation—Rates of protein breakdown
were measured by following the rate of tyrosine release into the medium. Because
muscle cannot synthesize or degrade tyrosine, its release reflects the rate of protein
breakdown. After a 1-h equilibration period, tissues were incubated for 2 h in fresh
medium of identical composition. Extensor digitorum longus pools of tyrosine did
not differ significantly in the presence or absence of IBMX (data not shown). In all
experiments, proteolysis was measured in the presence of cycloheximide
(0.5 mmol/L) into the incubation medium to prevent protein synthesis and
reincorporation of tyrosine back into proteins.
Hemodynamic parameters
Mean Arterial Pressure—The left carotid artery was cannulated and connected
to a pressure transducer (Braille Biomedical, Brazil) for measurements of mean
arterial pressure (MAP) that was recorded every 15 min throughout experiment.
Muscle Blood Flow—Blood flow changes around the microdialysis probe were
measured using the ethanol method described by Hickner et al. (15). Each
experiment included 90 min of perfusion with isotonic saline containing 0.5%
bovine albumin, 1 mmol/L glucose, 5 mmol/L ethanol in the presence or absence
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SHOCK JUNE 2007
INTERSTITIAL MUSCLE AMINO ACID
of 1 mmol/L IBMX or PTX. Dialysate samples were collected every 15 min
throughout experiment. Ethanol concentration was determined using a YSI 2700
select biochemical analyzer.
Determination of cAMP levels in muscles—The intracellular levels of cAMP
were measured in EDL muscles from sham-operated and septic rats treated or not
treated with IBMX (1 mmol/L) in vitro by using a method based on a competitive
enzyme immunoassay system (Amersham Biosciences). After 2 h of incubation (as
described above), muscles were homogenized in 6% trichloroacetic acid. After
extraction of lipid content with diethyl ether, the aqueous phase was lyophilized
and resuspended in the assay buffer.
TNF-! measurements—Plasma concentrations of TNF-! were determined by a
double-ligand enzyme-linked immunosorbent assay.
Calculations—The in vivo recovery of the microdialysis catheters was determined
in all samples and was calculated from the ratio of tyrosine concentration in the
dialysate/perfusate. To calibrate the catheters, [14C]-tyrosine (~2.500 desintegrations
per minute; 0.05 mmol/L) was added to the perfusate, and the fractional extraction of
radioactivity (percentage recovered at each period) was measured. The following
formula was used to calculate the interstitial tyrosine concentration:
½Tyrinterstitium ¼
½Tyrdialysate j½Tyrperfusate
recovery
þ ½Tyrdialysate
RESULTS
Relative recovery and muscle interstitial tyrosine levels in
normal rats—Table 1 shows the mean fractional outflux of
[14C]-tyrosine (relative recovery) through the microdialysis
catheter in skeletal muscle from normal rats. The calibration
method used herein has been validated in other microdialysis
studies (20) and predicts that the fractional outflux of the
labeled metabolite added in the perfusate is the same as its
relative recovery. Data show that the mean in vivo recovery of
tyrosine during the 3 different periods was 33 T 1%, and it did
not change along the experiment of microdialysis (Table 1).
This suggests that the present data were obtained in steadystate conditions throughout the study and were not affected by
artifacts such as the accumulation of the labeled tyrosine to
the microdialysis catheter. Like in other tissues, skeletal
muscle trauma caused by the catheter was small, as indicated
by histological examination and by the finding that muscle
glycogen store was not altered in dialyzed muscles as
compared with nondialyzed muscles (data not shown). The
relative recovery was decreased by CLP (20 T 1 vs. 34 T 1%
in sham operation; n = 18) but was not altered by cAMPphosphodiesterase inhibitors (data not shown).
TABLE 1. Muscle interstitial, arterial, interstitial-arterial tyrosine
concentration and fractional outflux of [14C]-tyrosine
(relative recovery) in normal rats during 80, 160, and
240 min of microdialysis
Time (min)
160
240
Interstitial tyrosine (nmol/mL)
85.3 T 3.7
91.3 T 8
95.6 T 9.7
Arterial tyrosine (nmol/mL)
57.0 T 2.8
56.3 T 3.3
58.3 T 2.9
Interstitial-Arterial tyrosine
(nmol/mL)
27.6 T 4.2
26.9 T 5.2
29.7 T 11
36 T 2
32 T 2
32 T 3
Fractional outflux (%)
Values are mean T SE of 10 animals.
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689
TABLE 2. Metabolic parameters, arterial blood gas levels, and HCT in
sham-operated and CLP rats
Sham
CLP
Glucose (mmol/L)
5.8 T 0.3
6.9 T 0.6
Lactate (mmol/L)
1.9 T 0.1
3.3 T 0.3*
1.2 T 0.01
0.15 T 0.01*
Muscle glycogen (%)
0.45 T 0.03
0.18 T 0.01*
pH
7.33 T 0.006
7.30 T 0.010
Liver glycogen (%)
PO2 (mmHg)
92 T 3
110 T 5*
PCO2 (mmHg)
37.3 T 1.6
20.1 T 0.9*
HCO3j (mmol/L)
19.3 T 0.7
9.7 T 0.7*
HCT (%)
42 T 2
47 T 1
Values are mean T SE from 7 animals.
* P G 0.05 vs. sham-operated rats.
HCT indicates hematocrit; PO2, arterial oxygen tension; PCO2, arterial
carbon dioxide tension; HCO3j, standard bicarbonate.
Changes in blood flow are expressed as ethanol outflow/inflow ratio, that is,
ethanol concentration in the dialysate/perfusate.
Statistical methods—Data are presented as mean T SE. Means from different
groups were analyzed using Student t test. Multiple comparisons were made by
using 1 or 2-way analysis of variance followed by Bonferroni t test. P G 0.05 was
taken as the criterion of significance.
80
IN
In the present study, the mean tyrosine concentration in
the skeletal muscle interstitial fluid from normal rats was
88 T 4 nmol/mL (Table 1). Similar results have been obtained
in the vastus lateralis muscle in humans (21, 22) and in
preliminary experiments from this laboratory in which tibialis
muscle from rats was perfused with a very slow rate of perfusion
(0.3 2L/min).
Characterization of the septic model—Sepsis was confirmed
by cultures of blood 3 h after CLP, which were positive for
aerobic and anaerobic bacteria. At this time, CLP rats
developed a marked lactate acidosis as evidenced by a
significant increase in the plasma concentration of lactate
and a decrease in the level of arterial plasma bicarbonate as
compared with sham-operated rats (Table 2). This was
compensated by alveolar hyperventilation in CLP rats, with
a significant decrease in PCO2 and increase in PO2. As
expected, CLP decreased liver and tibialis anterior muscle
glycogen content, but did not affect significantly plasma
levels of glucose (Table 2). Liver glycogen levels in shamoperated rats reported herein (Table 2) were lower than in
control nonoperated rats (4.5 T 0.3%; n = 9) probably due to
the surgical trauma of the animals. The wet/dry weights ratio
of tibialis anterior muscle in CLP (4.07 T 0.03 mg; n = 7) did
not differ from sham-operated rats (4.11 T 0.06 mg; n = 6).
Effect of sepsis on protein metabolism balance—As shown
in Figure 1, CLP induced a significant increase in muscle
interstitial tyrosine concentration (~150%), in arterial plasma
tyrosine levels (~50%), and in interstitial-arterial (I-A)
tyrosine concentration difference (~200%). These parameters
did not differ significantly between sham-operated and normal
rats (Table 1).
Systemic effect of PTX on protein metabolism balance and
plasma TNF-! levels—The intravenous PTX infusion
immediately after CLP induced a marked reduction of
tyrosine concentration in muscle interstitial fluid (~25%) and
in I-A difference (~50%), but it did not affect the arterial
plasma levels of tyrosine (Fig. 2B). The protein metabolism
balance in muscles from sham-operated rats was not altered
by PTX treatment (Fig. 2A). In sham-operated rats, plasma
levels of TNF-! averaged 8.4 T 4.6 pg/mL (n = 6). Three
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SHOCK VOL. 27, NO. 6
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ET AL.
FIG. 1. Interstitial, arterial plasma, and difference of I-A tyrosine
concentrations in sham-operated and CLP rats. The bars indicate the
values of tyrosine measured at the end of microdialysis experimental period
(3 h after CLP or sham surgery). Values are mean T SE of 9 rats. *P G 0.05
vs. sham-operated rats.
hours after CLP, TNF-! increased to 49.7 T 19 pg/mL (n = 6;
P G 0.05 compared with sham-operated). These values were
similar to those observed in previous studies using this model
of sepsis (23). Administration of PTX during 1 h after CLP
FIG. 3. In situ effect of IBMX on interstitial, arterial plasma, and
difference of I-A tyrosine concentrations in sham-operated (A) and CLP
rats (B). The bars indicate the values of tyrosine measured, at the end of
microdialysis experimental period (3 h after CLP or sham surgery), in animals
whose tibialis anterior muscles were perfused with saline containing or not
containing IBMX (10j3 M) during 90 min. Values are mean T SE of 8 rats.
*P G 0.05 vs. contralateral muscles perfused without IBMX.
FIG. 2. Systemic effect of PTX on interstitial, arterial plasma, and
difference of I-A tyrosine concentrations in sham-operated (A) and CLP
rats (B). The bars indicate the values of tyrosine measured, at the end of
microdialysis experimental period (3 h after CLP or sham surgery), in animals
previously treated with vehicle or PTX (50 mg/kg, i.v., during 1 h). Values are
mean T SE of 8 rats. *P G 0.05 vs. vehicle-treated rats.
suppressed the increase in plasma TNF-! concentration
(13.4 T 6.0 pg/mL, n = 5; P G 0.05 compared with nontreated
CLP rats).
In situ effects of cAMP-phosphodiesterase inhibitors on
protein metabolism balance—The addition of 10j3 M IBMX
to the perfusion medium reduced the interstitial tyrosine concentration (~35%) and the I-A difference (~45%) in muscles
from sham-operated (Fig. 3A) and septic rats (Fig. 3B), but it
did not affect the arterial plasma tyrosine. The concentration
of tyrosine in interstitium and arterial plasma was not altered
by perfusion with 10j3 M PTX (Fig. 4).
In vitro effects of IBMX on cAMP muscle levels—The
intracellular cAMP content did not differ significantly in
muscles from both groups (Fig. 5). Isobutylmethylxanthine
(10j3 M) in vitro increased the cAMP levels in muscles from
sham-operated (4-fold) and septic (3-fold) rats (Fig. 5).
Effect of sepsis and IBMX on the rate of overall proteolysis
in vitro—The addition of 10j3 M IBMX to the incubation
medium reduced the rate of tyrosine release by 32% and 35%
in sham-operated and CLP rats, respectively (Fig. 6). Lower
concentrations of IBMX (10j4 M) did not affect significantly
the rate of proteolysis in CLP group but reduced in 13% the
tyrosine release in muscles from sham-operated group (Fig. 6).
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SHOCK JUNE 2007
INTERSTITIAL MUSCLE AMINO ACID
IN
SEPSIS
691
FIG. 6. In vitro effect of IBMX at different concentrations on the rate
of proteolysis in EDL muscles from sham-operated and CLP rats. Three
hours after CLP or sham surgery, muscles were isolated and incubated for 2
h in the presence or absence of IBMX (10j5, 10j4 and 10j3 M). Data (mean T
SE of 7-9 muscle) are expressed as percentage of control rates, obtained in
the absence of IBMX. Rates of tyrosine release of control values for sham
and CLP averaged 0.309 T 0.009 and 0.376 T 0.017 nmol tyrosine I mg wet
weightj1 I 2 hj1, respectively. *P G 0.05 vs. contralateral muscles incubated
without IBMX.
FIG. 4. In situ effect of PTX on interstitial, arterial plasma, and
difference of I-A tyrosine concentrations in sham-operated (A) and CLP
rats (B). The bars indicate the values of tyrosine measured, at the end of
microdialysis experimental period (3 h after CLP or sham surgery), in animals
whose tibialis anterior muscles were perfused with saline containing or not
containing PTX (10j3 M) during 90 min. Values are mean T SE of 8 rats.
At the lowest concentration (10j5 M), IBMX did not affect
muscle tyrosine release either in sham-operated or in CLP rats
(Fig. 6). The rate of overall proteolysis in EDL muscles from
rats submitted to CLP was 20% higher than in sham-operated
FIG. 5. Intracellular levels of cAMP in EDL muscles from shamoperated and CLP rats incubated in the presence of IBMX. Three hours
after CLP or sham surgery, muscles were isolated and incubated for 2 h in
the presence or absence of IBMX (10j3 M). Values are mean T SE of 8
muscles. *P G 0.05 vs. sham-operated muscles incubated with IBMX. † P G
0.05 vs. contralateral muscles incubated without IBMX.
rats (Fig. 7). As shown in Figure 7, the addition of H89
completely blocked the antiproteolytic effect of 10j3 M
IBMX on both muscles from sham-operated and septic rats.
Previous experiments showed that proteolysis was not
affected by the isolated addition to the incubation medium
of H89 at the dose used (data not shown).
Hemodynamic parameters—The MAP was progressively
decreased in rats undergoing CLP compared with shamoperated rats during all the experimental periods (Fig. 8).
Relative changes in MBF, calculated from the ethanol
outflow/inflow concentration ratio, were increased. As shown
in Figure 9, this ratio was increased (~30%) in CLP rats up to
60 min of the beginning of microdialysis, indicating a
reduction of MBF induced by sepsis. IBMX in situ did not
affect MBF either in sham-operated or in septic rats (Fig. 9).
DISCUSSION
The release of tyrosine from rat skeletal muscle was
monitored indirectly by measurements of this amino acid in
the interstitial fluid after 3 h of CLP. Cecal ligation with 2
punctures in rats provides a satisfactory model for studying
the acute catabolic states. Because most animals died 16 to 24
h after CLP, the present model does represent a rapidly lethal
model of sepsis in which it is possible to study pathological
alterations in different tissues, including skeletal muscle
(reviewed in (24)). In the present microdialysis study, CLP
resulted in a drastic increase in the I-A tyrosine concentration
difference, indicating net protein degradation in tibialis
anterior muscle in vivo (Fig. 1). Although the circulatory
changes induced by sepsis may have overestimated the values
of tyrosine concentration found in the interstitium, a change in
blood flow is not likely to be a major cause of the effects
observed in skeletal muscle from septic rats. This contention
is supported by previous findings that a comparable decrease
in blood flow as noted with CLP (i.e., j30%) did not increase
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692
SHOCK VOL. 27, NO. 6
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ET AL.
FIG. 9. Effect of CLP and IBMX perfusion on ethanol outflow/inflow
concentration ratio in skeletal muscle. Microdialysis catheters were
inserted into tibialis anterior muscles of CLP or sham-operated rats and
perfused with saline containing ethanol (5 mmol/L) supplemented or not with
IBMX (10j3 M) during 90 min. Values are mean T SE of 9 rats. *P G 0.05 for
CLP vs. sham-operated rats.
FIG. 7. Rates of proteolysis in EDL muscles from sham-operated (A)
and CLP rats (B) incubated in the presence of IBMX (10j3 M) and H89
(50 2M). Three hours after CLP or sham surgery, muscles were isolated and
incubated for 2 h in the presence or absence of drugs. Values are mean T SE
of 7 muscles. *P G 0.05 vs. muscles incubated without IBMX or H89. † P G
0.05 vs. muscles incubated with IBMX. #P G 0.05 vs. sham-operated rats.
the interstitial tyrosine concentration in gastrocnemius
muscles from normal rats perfused with !-adrenergic agonists
and vasopressin (25).
FIG. 8. Mean arterial blood pressure in sham-operated and CLP rats
during the microdialysis experimental period. Values are mean T SE of
10 rats. *P G 0.05 vs. sham-operated rats.
The present in vitro experiments show that the rate of
tyrosine release (an index of proteolysis) in EDL muscle was
increased in CLP (Fig. 7) rats, suggesting that the muscle
interstitial tyrosine increase observed in vivo was probably
because of the activation of proteolytic pathways. This finding
is consistent with the increase in protein degradative machinery reported in septic animals and patients (reviewed in (26)).
A concomitant decrease in the rate of protein synthesis or
uptake of amino acid that have been shown in rat skeletal
muscle 16 h after CLP cannot be ruled out (27, 28). It might
be argued that the reduced volume of the interstitium, rather
than net protein catabolism, might have contributed to the
increased tyrosine concentrations found in interstitial fluid of
septic rats. However, it should be noted that every microdialysis catheter was individually calibrated in situ, and
because the recovery marker was constant (Table 1), eventual
volume changes in the interstitium were corrected. Although
the interstitial volume has not been directly measured in the
present study, the findings that hematocrit (Table 2) and the
wet/dry muscle weights ratio (BResults^) in CLP rats were not
different from sham-operated rats suggest that the 3-h sepsis
did not result in muscle dehydration. Indeed, it has recently
been shown in patients with sepsis that the efflux of amino
acids from muscle into the interstitium is due to alterations in
the rate of net muscle protein catabolism and is not likely
related to any alterations in tissue volume or MBF (22).
The main systems involved with the sepsis-induced increase
in muscle protein breakdown are the Ca2+ and ubiquitin
(Ub)Yproteasome-dependent pathways (26). The former
releases myofilaments from the sarcomere in an early ratelimiting component of this catabolic response in muscle. The
released myofilaments are ubiquitinated and degraded by the
26S proteasome (26). Previous studies have shown that both
PTX and torbafylline treatment for 9 days suppress the increased
expression of different components of Ub-proteasome proteolytic pathway by inhibiting the hyperproduction of TNF-! in
Copyright @ 2007 by the Shock Society. Unauthorized reproduction of this article is prohibited.
SHOCK JUNE 2007
cancer and septic rats (10, 11). We further show that a single
intravenous injection of PTX immediately after CLP prevented the plasma TNF-! increase and significantly reduced
by 50% the I-A tyrosine concentration difference in septic rats
(Fig. 2B), indicating a rapid anticatabolic effect of PTX on
skeletal muscle protein metabolism. The fact that PTX
treatment did not affect tyrosine levels in muscles from
control healthy animals (Fig. 2A) suggests that this drug
reduces protein catabolism indirectly by suppressing high
levels of TNF-! in septic rats. This contention is supported
by the finding that tyrosine interstitial concentration in
muscles from both CLP and sham-operated rats was not
altered by in situ perfusion with PTX (Fig. 4). Nevertheless,
an alternative hypothesis is that PTX reduces muscle protein
catabolism directly by increasing cAMP intracellular levels in
skeletal muscle. Interestingly, Hinkle et al. (9) has recently
shown that rolipram, a selective phosphodiesterase 4 inhibitor,
decreases the loss of skeletal muscle mass and function in two
disuse atrophy models (casting and denervation) without a
cytokine-mediated mechanism. These authors hypothesized
that cAMP-phosphodiesterase inhibitors might have direct
effects on skeletal muscle inhibiting proteolysis, an inference
that is supported by the present data showing that addition of
IBMX to the perfusion medium reduced by 45% the I-A
tyrosine concentration difference in muscle from shamoperated rats (Fig. 3A) without changes in MBF (Fig. 9).
It is noteworthy that the anticatabolic effect induced by
10j3 M IBMX perfusion in vivo was quite similar to the
antiproteolytic effect seen in EDL muscles incubated in the
presence of this compound in vitro (Fig. 6). Similar in vitro
findings have been previously reported in soleus muscles from
normal rats (5) and in chicks (29). The present data also show
that 10j3 M IBMX in situ and in vitro reduces the increased
muscle protein catabolism in septic rats, and that its anticatabolic effect in vitro is less effective in septic than in shamoperated rats because the latter did not respond to lower
concentrations of IBMX (10j4 M; Fig. 6). The finding in this
study that IBMX reduced basal levels of proteolysis rather than
prevented the sepsis-induced increase in proteolysis may be
accounted for by differences in the activity of cyclic nucleotide
phosphodiesterase, which has been shown to be stimulated by
TNF-!, resulting in a cAMP fall in endothelial cells (30).
Although the basal levels of cAMP was not different in
muscles from sham and CLP rats, the IBMX-induced increase
in cAMP muscle levels was smaller in EDL from CLP rats as
compared with sham (Fig. 5). Further experiments are needed
to certify if the activity of cAMP-phosphodiesterase is also
increased in skeletal muscles from acutely septic rats.
Taken together, these data strongly suggest that the inhibitory action of IBMX on skeletal muscle proteolysis is direct
and mediated by cAMP. The findings that activation of the
guanosine triphosphateYbinding protein stimulatoryYcoupled
"-adrenergic receptor in vitro inhibits proteolysis in skeletal
muscle from normal rats (5) and reduces skeletal muscle
atrophy in vivo (31) support this hypothesis. The antiproteolytic effect of IBMX in vitro was inhibited by H89, a selective
PKA inhibitor (Fig. 7), further supporting the idea that
activation of the cAMP-dependent pathway via PKA is one
INTERSTITIAL MUSCLE AMINO ACID
IN
SEPSIS
693
of the regulatory mechanism(s) to prevent excessive breakdown of proteins in skeletal muscle. Although the available
data do not allow any conclusion about the identity of these
systems, we have recently provided evidence for the existence
of an inhibitory adrenergic tonus in skeletal muscle that
restrains proteolysis by keeping the Ca2+-dependent pathway
inhibited (4). A close association between adrenergic activity
and Ca2+-dependent proteolysis has also been obtained in
numerous studies, showing that the activity and gene
expression of 2-calpain and calpastatin, its endogenous
inhibitor, are decreased and increased, respectively, after "adrenergic agonist treatment (32). Because catecholamines
and "2-adrenergic agonists activate cAMP-dependent PKA in
rat skeletal muscle (33), it has been proposed that both
calpastatin and calpains are targets for this kinase. In fact,
evidence in fibroblasts indicates that m-calpain can be directly
phosphorylated by PKA, and that the epidermal growth
factorYinduced calpain activity is suppressed (34). That the
UbYproteasome-dependent proteolysis may also be inhibited
by cAMP-dependent pathways is suggested by studies showing that the hyperactivation of this system that occurs in
skeletal muscle of tumor-bearing rats is effectively reduced by
clenbuterol (31) and PTX (35) treatment. The in vitro
inhibition of Ca2+-dependent and UbYproteasome-dependent
proteolysis by PTX observed in isolated muscles from normal
and diabetic rats (A. Baviera, unpublished observation) (36) is
consistent with the above studies.
In summary, the present work shows that pharmacological
inhibition of cAMP-phosphodiesterase reduces skeletal
muscle protein catabolism in vivo and in vitro. Pentoxifylline
exerts these effects indirectly by suppressing high levels of
TNF-! in septic rats. The anticatabolic effect of IBMX on
skeletal muscle is direct, suggesting the participation of
cAMP-dependent intracellular pathways and PKA in the
inhibitory control of skeletal muscle proteolysis. This finding
may be helpful for the treatment of skeletal muscle atrophy in
different wasting muscle conditions.
ACKNOWLEDGMENTS
This study was supported by the Funda0ão de Amparo à Pesquisa do Estado de
São Paulo (grant no. Fapesp 04/02674-0) and São José do Rio Preto Medical
School (grant no. BAP 1893/05). During this study, E.C.L. received a fellowship
from Coordena0ão de Aperfei0oamento de Pessoal de Nı́vel Superior (CAPES),
and A.B.M. received a fellowship from Conselho Nacional de Pesquisa (CNPq).
We are indebted to Dr. Fernando Cunha and Giuliana Bertozi for TNF-!
measurements.
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