Kaohsiung Medical University Institutional Repository:Item 310902000/10547

G PROTEIN AND ADENYLATE CYCLASE COMPLEX-MEDIATED SIGNAL

TRANSDUCTION IN THE RAT HEART DURING SEPSIS

Li-Ling Wu,* Shaw-Lang Yang,

Rei-Cheng Yang,

Hseng-Kuang Hsu,

Chin Hsu,

Lin-Wang Dong,

and Maw-Shung Liu

*Department of Physiology and Pathophysiology, Peking University Health Science Center, China;

Department of Physiology, Kaohsiung Medical University, Kaohsiung, Taiwan; and

Department of

Pharmacological and Physiological Science, Saint Louis University School of Medicine,

St. Louis, Missouri

Received 20 Sep 2002; first review completed 18 Oct 2002; accepted in final form 19 Dec 2002

ABSTRACT—Changes in the protein level of various subunits of GTP-binding protein and the activity of adenylate cyclase in the rat heart during different phases of sepsis were studied. Sepsis was induced by cecal ligation and puncture (CLP). Experiments were divided into three groups: control, early sepsis, and late sepsis. Early and late sepsis refers to those animals sacrificed at 9 and 18 h, respectively, after CLP. The protein levels of various subunits of GTP-binding protein were determined by Western blot analysis. The activity of adenylate cyclase was measured based on the rate of formation of cAMP from [␣-32_{P]ATP. The results show that protein levels of G␣s and G␤remained stable during the early and the}

late phases of sepsis. The protein levels of G␣i-2 and G␣i-3 remained relatively unaltered during the early phase of sepsis, but they were increased by 46.5% (P < 0.05) and 61.3% (P < 0.01), respectively, during the late phase of sepsis. The basal adenylate cyclase activity remained unchanged during the early phase while it was decreased by 25.7% (P < 0.05) during the late phase of sepsis. The isoproterenol-stimulated adenylate cyclase activity was unchanged during early sepsis while it was decreased by 44.6% (P < 0.01) during late sepsis. These data demonstrate that during the late hypodynamic phase of sepsis, myocardial G␣i-2 and G␣i-3 protein levels were increased and the increases were coupled with a reduction in adenylate cyclase activity. Because GTP-binding proteins mediate sympathetic control of cardiac function, the present findings may have a pathophysiological significance in contributing to the understanding of the pathogenesis of cardiac dysfunction during the late stage of sepsis.

KEYWORDS—GTP-binding proteins, signal transduction, septic shock, myocardial dysfunction, heart failure

INTRODUCTION

Sympathetic control of cardiac function is mediated through

GTP-binding proteins. GTP-binding proteins are composed of

a large family of highly homologous proteins that couple more

than 100 different receptors with different effector enzymes

(1–3).

␤

-adrenergic receptor (

␤

AR;

␤

AR and

␤

AR) and

␣

-adrenergic receptor (

␣

AR;

␣

AR) are coupled to G

␣

s and

Gq subunits, respectively, whereas

␣

AR is coupled to G

␣

i

subunit of GTP-binding proteins (1, 4–6). There is direct

evidence that organ-specific alterations in GTP-binding protein

expression contribute to the pathogenesis of heart diseases in

human and animal models. Changes in G

␣

s and G

␣

i protein

levels and the gene transcripts encoding them are found to be

associated with heart failure, ischemic cardiomyopathy,

hyper-trophy, and hypertension (7–13). In shock and sepsis, G

␣

i

levels are found to be elevated in the heart of

catecholamine-refractory septic shock patients (14) and in the neonatal rat

heart myocytes upon exposure with the plasma of

noradrena-line-treated septic shock patients (15). Because the available

data in the literature regarding changes in myocardial

GTP-binding proteins are limited to the hypodynamic phase and

because sepsis is a two-phase process in which patients initially

go through a hyperdynamic phase and, subsequently, the

hypo-dynamic phase, it is important that an animal model capable of

exhibiting a biphasic septic syndrome be adapted for the

inves-tigation of the role of GTP-binding proteins in the pathogenesis

of heart dysfunction in septic shock. Furthermore, because

previous work from this laboratory has revealed that

␤

AR and

␣

AR undergo a biphasic expression in the rat heart during the

two distinct cardiodynamic phases of sepsis (16–18), the

present work dealing with altered G protein/adenylate cyclase

system was undertaken to clarify changes in postreceptor

events in an attempt to understand the pathogenesis of

myocar-dial dysfunction during the progression of sepsis.

MATERIALS AND METHODS

Animal model

All animal experiments in this study were performed with the approval of the Animal Care Committee of Saint Louis University School of Medicine and in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Male Sprague-Dawley rats weighing from 270–320 g were used. All animals were fasted overnight with free access to water before the induc-tion of sepsis. Sepsis was induced by cecal ligainduc-tion and puncture (CLP) as described by Wichterman et al. (19) with minor modifications (20). Under halothane anes-thesia, a laparotomy was performed (the size of the incision was 2.5 cm), and the cecum was ligated with a 3-0 silk ligature and punctured twice with an 18-gauge needle. The cecum was then returned to the peritoneal cavity and the abdomen was closed in two layers. Control rats were sham operated (a laparotomy was performed and the cecum was manipulated but neither ligated nor punctured). It should be mentioned that the values of the biochemical and the molecular biological indexes (GTP-binding protein levels and adenylate cyclase activities) that we reported in this study were indistinguishable between the sham-CLP and the nonsurgical controls. All animals were resuscitated subcutaneously with 4 mL of 0.9% NaC1/100 g body weight at the completion of surgery and also at 7 h postsurgery. Animals were fasted but had free access to water after operative procedure. Experiments were divided into three groups: control, early sepsis, and late sepsis. Early and late sepsis refers to those animals sacrificed at 9 and 18 h, respectively, after CLP. Previous experi-Address reprint requests to Dr. Rei-Cheng Yang, Department of Physiology,

Kaohsiung Medical University, Kaohsiung, Taiwan.

This work was supported by the National Heart, Lung and Blood Institute (grant no. HL-30080) and the National Institute of General Medical Sciences (grant no. GM-31664), the National Institutes of Health.

DOI: 10.1097/01.shk.0000055816.40894.cd

ments have indicated that myocardium was in the hyperdynamic state during the early phase, whereas it was in the hypodynamic state during the late phase of sepsis (18). The mortality rates were 0% for control, 4% for early sepsis, and 27% for late sepsis.

Preparation of cardiac sarcolemmal membranes

Rat heart sarcolemmal membranes were prepared according to a procedure previously used in this laboratory (16, 17). Hearts removed from control and septic rats were weighed, freeze-clamped with aluminum clamps precooled in liquid nitro-gen, and pulverized with a mortar and pestle precooled in liquid nitrogen. The powdered tissues were thawed and homogenized with Tekmar Tissumizer (Model SDT) in 5 vol of buffer A (50 mM NaH2PO4/Na4P2O7, pH 7.4; 10 mM EDTA; 25 mM NaF; 1␮g/mL soybean trypsin inhibitor; 1 ␮g/mL aprotinin; 0.75 ␮g/mL pepstatin A; and 2␮g/mL leupeptin). The homogenates were centrifuged at 14,000 g for 20 min. The resulting pellets were suspended in buffer B (0.6 M NaCl, 50 mM NaH2PO4/Na4P2O7, pH 7.4; 10 mM EDTA; and 25 mM NaF), rehomogenized, and recentrifuged at 14,000 g for 20 min. The pellets from the second centrifugation were suspended in buffer A, homogenized three times (each time for 30 s), and centrifuged at 2300 g for 15 min. The resulting pellets were resuspended, rehomog-enized, and recentrifuged at 2300 g for 15 min. All homogenizations throughout were performed with Tekmar Tissumizer. The 2300-g supernatants were combined and centrifuged at 79,700 g for 40 min. The 79,700-g pellets were suspended in 1 M sucrose dissolved in buffer C (0.3 M NaCl, 50 mM NaH2PO4/Na4P2O7, and 100 mM Tris-HCl, pH 7.4). Ten milliliters of this suspension were layered at the bottom of a discontinuous sucrose gradient consisting of 9 mL of 0.6 M sucrose (dissolved in buffer C) and 9 mL of 0.25 M sucrose (dissolved in 10 mM histidine, pH 7.4). The gradients were centrifuged at 254,000 g for 70 min. Fractions at 0.6:0.25 M sucrose interfaces were collected, diluted with buffer C, and then centrifuged at 162,600 g for 40 min. The final pellets were suspended in a buffer containing 0.25 M sucrose and 30 mM histidine (pH 7.4), stored at −80°C, and then used as sarcolemmal membranes. The entire procedure was performed at 4°C unless other-wise stated.

Determination of GTP-binding protein levels by Western

blot analysis

Western blot analysis was performed according to the method of Ausubel et al. (21) with minor modification (22). Sarcolemmal membranes containing various amounts of protein (100␮g for G␣s, 100 ␮g for G␣i-2, 100 ␮g for G␣i-3, and 40 ␮g for ␤-subunit) were denatured and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10–20% sulfate-polyacrylamide gradient gel). Using the Bio-Rad Mini Trans-Blot Electrophoretic Transfer Cell, proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were transferred to a polyvinylidene fluoride membrane (Bio-Rad) at 8 mA (constant current) for 14–16 h in transfer buffer (25 mM Tris base, pH 8.3; 192 mM glycine; 5% [vol/vol] methanol). Nonspecific binding sites were blocked with 10% (wt/vol) of nonfat dry milk in Tris-buffered saline (TBS: 20 mM Tris-HC1, pH 7.4; 137 mM NaC1) for 4 h at 4°C. Blots were washed three times (10 min each) with TBST (TBS containing 0.5% Tween-20) followed by incubation with a 1:1000 dilution of a polyclonal antibody recognizing G␣s, G␣i-1 and G␣i-2, G␣i-3, or G␤ subunit (Calbiochem-Novabiochem International, San Diego, CA) for 5 h at room temperature. Subse-quently, the blots were washed three times (10 min each) with TBS and then incubated with a 1:2,000 dilution of an antirabbit immunoglobulin, peroxidase-linked species-specific whole antibody (Amersham Life Science) for 1 h at room temperature. The blots were then washed four times (10 min each) with TBST, followed by incubation with enhanced chemiluminescent (ECL) Western blotting detection reagent (Amersham Life Science), and finally exposed to Hyperfilm-ECL (Amersham Life Science) for 40 s. Autoradiographs were scanned with Hewlett-Packard ScanJet 4C Scanner, and the relative densities were quantified by Jandel Scientific Software program (Sigma Gel). It should be mentioned that the quanti-tation of immunoblots was performed by comparing each individual subunit of GTP-binding protein to the control only for that particular blot. For each subunit, six separate blots were performed. Each blot consisted of control, early septic, and late septic samples in equal amount of protein. Preliminary experiments have indicated that the control levels (six control samples prepared from six different animals and performed on the same blot) for each subunit were almost identical.

Assay of adenylate cyclase activity

Adenylate cyclase activity was determined by a method previously used in this laboratory (22) with modification. The reaction mixture in a final vol of 200␮L contained 50 mM Tris-HC1, pH 7.5; 5 mM MgCl2; 5 mM creatine phosphate; 0.4 mg/mL creatine phosphokinase; 1 mM cAMP; 1 mM dithiothreitol; 1 mM [␣-32_{P]ATP (2 × 10}6_{counts/min). The reaction was initiated by the addition of} sarcolemmal membranes containing 100␮g of protein and was allowed to proceed for 8 min at 37°C. The reaction was terminated by the addition of 0.2 mL of a solution containing 2% sodium dodecyl sulfate; 40 mM ATP; 50 mM Tris-HCL, pH 7.5; 1.4 mM [2,8-3_{H]cAMP (20,000 counts/min). The reaction mixture was then} diluted with 0.6 mL of deionized water and was decanted into Dowex columns. The eluate from this and two subsequent 1-mL washes was discarded. Three milliliters of water were then added to each column and the eluate was collected in a test tube containing 0.2 mL of 1.5 M imidazole (pH 7.5). After mixing, the contents of each tube were poured into a second column containing neutral alumina pre-equilibrated with 0.1 M imidazole HC1 (pH 7.5). The eluate of the alumina column was collected directly into a scintillation vial and then counted by a liquid scintillation counter. It should be mentioned that [2,8-3

H]cAMP was included in the stop solution to deter-mine the recovery of cAMP from the columns.

Other assays

(Na+_-K+_{)-ATPase, Ca}2+_{-ATPase, and glucose-6-phosphatase activities were} determined as described previously by us (22). The protein concentration of sarco-lemmal membranes was determined by the method of Lowry et al. (23).

Statistical analysis

The statistical analysis of the data was performed using one-way ANOVA followed by Student-Newman-Keuls tests. A P value of less than 0.05 was accepted as statistically significant.

Materials

[␣-32_{P]ATP (650 Ci/mmol) and [2,8-}3_{H]adenosine-3⬘,5⬘-cyclic monophosphate} (38 Ci/mmol) were purchased from ICN Pharmaceuticals, Inc. Horseradish peroxi-dase-labeled second antibody conjugate (donkey anti-rabbit), ECL Western blotting detection reagent, and Hyperfilm were obtained from Amersham Life Science. ATP, cyclic AMP, creatine phosphate, and creatine phosphokinase were purchased from Sigma Chemical Co. Purified primary antibodies, including rabbit anti-G␣s subunit, C-terminal (385-394); rabbit anti-G␣i-1 and G␣i-2 subunits, C-terminal (345-354); rabbit anti-G␣i-3 subunit, C-terminal (345-354); and rabbit anti-G-protein ␤ subunit, internal (127–139) were supplied by Calbiochem-Novabiochem Interna-tional. Other chemicals and reagents were of analytical grade.

RESULTS

Table 1 shows marker enzyme activities of heart

sarcolem-mal membranes isolated from control and septic rats. (Na

-K

_{)-ATPase served as marker for sarcolemmal membranes,}

while Ca

_{-ATPase and glucose-6-phosphatase served as}

markers for sarcoplasmic reticulum. In sarcolemmal

membranes, (Na

-K

)-ATPase activities were enriched 17- to

19-fold whereas Ca

-ATPase and glucose-6-phosphatase

activities were not enriched as compared with the homogenates

within each experimental group. The pattern of changes in the

enzymatic marker in the sarcolemmal membrane fraction was

essentially identical among the three experimental groups:

control, early sepsis, and late sepsis. These data indicate that

sarcolemmal membrane preparations were highly purified and

minimally contaminated with sarcoplasmic reticulum.

Further-more, the degrees of purity of sarcolemmal membranes and the

extents of cross contamination with sarcoplasmic reticulum

Control Early sepsis Late sepsis

Homo SL Homo SL Homo SL

(Na+_{+ K}+_{)-ATPase 1.5 ± 0.2 29.0 ± 1.4 1.7 ± 0.4 28.1 ± 0.9 1.4 ± 0.2 24.0 ± 1.7}

Ca2+_{-ATPase 2.4 ± 0.7 3.3 ± 0.4 2.1 ± 0.6 3.2 ± 0.3 2.7 ± 0.9 3.1 ± 0.2}

Glucose-6-phosphatase 2.5 ± 0.6 2.4 ± 0.4 2.1 ± 0.3 3.1 ± 0.2 2.1 ± 0.3 3.3 ± 0.2 Values are Mean ± SE in µmol/mg/h. Each value represents a mean of six experiments. Homo, homogenate; SL, sarcolemmal membrane.

among the control, the early septic and the late septic groups

were the same.

Preliminary experiments were conducted to correlate

autora-diographic intensities of the immunoblots with various amounts

of GTP-binding protein (G

␣

s, G

␣

i-2, G

␣

i-3, and G

␤

) in the

control rats before the full-scale Western blot analysis. The values

of signal intensity obtained were proportional to the amounts of

protein in the putative GTP-binding protein subspecies bands

(data not shown). Based on these observations, appropriate

concentrations of sarcolemmal membrane proteins within linear

range were then selected (100

␮

g for G

␣

s, 100

␮

g for G

␣

i-2, 100

␮

g for G

␣

i-3, and 40

␮

g for

␤

subunit) for Western blot analysis

for the comparison between control and septic experiments.

Figure 1 shows the Western blot analysis of G

␣

s protein

levels in rat heart sarcolemmal membranes during different

phases of sepsis. Two putative bands for G

␣

s, with molecular

weights of 45 and 42 kDa, were detected. This finding is

consistent with that reported in the literature (24). Quantitative

analysis of the data reveals that the protein levels for 45-kDa

and 42-kDa forms of G

␣

s were unaltered during both early and

late phases of sepsis. These findings demonstrate that G

␣

s

protein levels remained unaffected in rat heart sarcolemmal

membranes during the progression of sepsis.

Figures 2 and 3 depict changes in G

␣

i-2 and G

␣

i-3 protein

levels in rat heart sarcolemmal membranes during the early and

the late phases of sepsis. Western blot analysis reveals that

G

␣

i-2 protein levels were not significantly affected (+17.3%; P

> 0.05) during the early phase but were increased by 46.5% (P

< 0.05) during the late phase of sepsis (Fig. 2). It should be

noted that the anti-G

␣

i-2 antibody used in this study cross

reacts with G

␣

i-l species. The fact that G

␣

i-l signal was not

detected in our study indicates that G

␣

i-l was absent from rat

heart sarcolemmal membranes (25). As shown in Figure 3,

FIG. 1. Western blot analysis of G␣s protein level in the sarcolemmal

membranes of control and septic rat hearts. Western blot analysis was

performed as described under the Materials and Methods section using poly-clonal antibody raised specifically against the G␣s subunits. Vertical bars indicate SEM. Number of experiments is shown in the parenthesis within each column. C, Control; ES, early sepsis; LS, late sepsis.

FIG. 2. Western blot analysis of G␣i-2 protein level in the

sarcolem-mal membranes of control and septic rat hearts. Western blot analysis

was carried out as described under the Materials and Methods section using polyclonal antibodies raised specifically against the G␣i-1 and G␣i-2 subunits of G-protein. The arrow shows the molecular weight of G␣i-2 subunit. Vertical bars indicate SEM. Number of experiments is shown in the parenthesis within each column. C, Control; ES, early sepsis; LS, late sepsis.

FIG. 3. Western blot analysis of G␣i-3 protein level in the

sarcolem-mal membranes of control and septic rat hearts. Western blot analysis

was performed as described under the Materials and Methods section using polyclonal antibody raised specifically against the G␣i-3 subunit of G-protein. The arrow indicates the molecular weight of G␣i-3 subunit. Vertical bars indicated SEM. Number of experiments is shown in the parenthesis of each column. C, control; ES, early sepsis, LS, late sepsis.

G

␣

i-3 protein levels remained relatively stable (+11.4%, P >

0.05) during early sepsis whereas they were increased by

61.3% (P < 0.01) during late sepsis (Fig. 3). The data presented

in Figures 2 and 3 demonstrate that G

␣

i-2 and G

␣

i-3 protein

levels were increased during the late stage of sepsis.

Figure 4 shows the Western blot analysis of G

␤

protein

levels in rat heart sarcolemmal membranes during the progress

of sepsis. Quantitative analysis of Western blot indicates that

G

␤

protein levels remained unaltered during the early and late

phases of sepsis. It should be mentioned that the G

␤

antibody

used in this study cross reacts with both

␤

1 and

␤

2 subunits of

GTP-binding protein. In our experiments, only

␤

2 subunit with

a molecular weight of 36 kDa was detected, indicating that

␤

2

is the predominant subtype of G

␤

expressed in the myocardium

(26). These findings indicate that G

␤

protein level was

unaf-fected during the progression of sepsis.

Figure 5 depicts changes of adenylate cyclase activity in rat

heart sarcolemmal membranes during the two

cardiodynami-cally distinct phases of sepsis. Basal adenylate cyclase activity

remained unchanged during the early phase whereas it was

decreased by 25.7% (P < 0.05) during the late phase of sepsis

(Fig. 5A). Similarly, the isoproterenol-stimulated adenylate

cyclase activity was unchanged during early sepsis while it was

decreased by 44.6% (P < 0.01) during late sepsis (Fig. 5B).

These data indicate that the functional endpoint for

␣

-subunit

of GTP-binding protein, the adenylate cyclase activity, was

decreased during the late hypodynamic phase of sepsis.

DISCUSSION

GTP-binding proteins mediate sympathetic control of

cardiac function. The best characterized GTP-binding proteins

consist of two major classes: the stimulatory component, Gs,

which stimulates and the inhibitory component, Gi, which

inhibits adenylate cyclase activity (27). Adenylate cyclase is a

membrane-bond enzyme that catalyzes the hydrolysis of ATP

to cAMP. cAMP, an intracellular messenger, activates protein

kinase A and then initiates a series of enzymatic reactions

leading to a phosphorylation cascade of multiple proteins that

eventually regulate both the rate and the force of cardiac

contraction (27). In the heart, catecholamines released into the

synaptic cleft at sympathetic nerve terminals bind to either

␣

AR or

␤

AR on the cardiac sarcolemma. Stimulation of

␤

AR,

␤

AR, and

␣

AR activates G

␣

s and leads to increased cAMP

production through adenylate cyclase activation, whereas

stimulation of

␣

AR activates G

␣

i and leads to decreased

cAMP production through adenylate cyclase inactivation (1,

27). An increase or a decrease in G

␣

s and/or G

␣

i levels

coupled with activation or inactivation of adenylate cyclase,

thus function as a modulating system regulating myocardial

contractility. The present study in cardiac sarcolemma reveals

that during late sepsis, the G

␣

i-2 and G

␣

i-3 protein levels were

increased and the increases were coupled with a decrease in

adenylate cyclase activity, may have a pathophysiological

significance in contributing to the understanding of the

patho-genesis of cardiac dysfunction during the late stage of sepsis.

The exact mechanisms responsible for the increase in G

␣

i-2

and G

␣

i-3 protein levels coupled with a decrease in adenylate

cyclase activity were not completely understood. It has been

reported that in the rat, a prolonged infusion of isoprenaline

decreased myocardial

␤

AR number and increased heart G

␣

i

content (28, 29). In rat heart muscle cells, exposure to

noradrenaline decreased

␤

AR density, increased G

␣

i-2 and

G

␣

i-3 protein levels, and reduced adenylate cyclase sensitivity

(30). The noradrenaline-induced increase in Gi protein

␣

-sub-units and adenylate cyclase desensitization were blocked by a

␤

AR antagonist (30). In human end-stage heart failure,

myocardial

␤

AR density was decreased and the decrease in

␤

AR density was accompanied by an increase in G

␣

i protein

content (7, 12, 31). These reports demonstrate that a decreased

␤

AR density in response to an enhanced sympathetic drive is

an important causative factor leading to the increased G

␣

i

protein level and the decreased adenylate cyclase sensitivity.

membranes of control and septic rat hearts. Western blot analysis was

carried out as described under the Materials and Methods section using polyclonal antibody raised specifically against the␤ subunit of G-protein. The arrow shows the molecular weight of G␤ subunit. Vertical bars indicate SEM. Number of experiments is shown in the parenthesis within each column. C, Control; ES, early sepsis; LS, late sepsis.

FIG. 5. Changes in adenylate cyclase activities in the rat heart during

different phases of sepsis. Adenylate cyclase activities were assayed as

described under the Materials and Methods section except that for isoproterenol stimulated adenylate cyclase (B), 1 mM of isoproterenol was present in the reaction mixture. Vertical bars indicate SEM. Number of experiments is shown in the parenthesis within each column. C, Control; ES, early sepsis; LS, late sepsis.

The fact that myocardial

␤

AR and

␣

AR densities have been

found to greatly reduced in response to the elevated circulating

catecholamines during the late phase of sepsis (16–18), it is

conceivable that the observed increase in the G

␣

i-2 and G

␣

i-3

protein levels and the decrease in the adenylate cyclase activity

are the results of underexpression of

␤

AR and

␣

AR. In

addi-tion to the altered

␤

AR and

␣

AR dynamics, changes in

aden-osine A1 receptor may contribute to the increase in G

␣

i protein

level and the decrease in adenylate cyclase activity because

adenosine A1 receptor is known to exist in cardiac myocytes

which coupled to G

␣

i and cyclase inhibition (32). Further

study is required to clarify role of adenosine A1 receptor on the

altered G

␣

i and cyclase inhibition in the heart during sepsis.

Because receptors and their coupling proteins belong to

differ-ent groups of genes that are controlled by distinct mechanisms of

regulation (33), it is possible that the altered expression of various

subspecies of GTP-binding protein is a result of modification of

their gene transcripts, instead of a secondary response to the

altered receptor dynamics. In heart failure, plasma catecholamine

levels were elevated and the elevated plasma catecholamine was

correlated with an increase in the myocardial G

␣

i-2 mRNA

abun-dance and an unchanged G

␣

s mRNA level (7, 12). It is of interest

to note that a consensus sequence of a “cAMP response element”

has been described in the promoter region of the G

␣

i-2 gene (34),

whereas it is lacking in the G

␣

s gene (35). These findings suggest

that an increased sympathetic drive via the cAMP cascade would

lead to an increased transcription rate of the G

␣

i-2, but not that of

the G

␣

s genes. Whether a similar transcriptional regulatory

mechanism is responsible for the increase in G

␣

i-2 and G

␣

i-3,

but not the G

␣

s, protein levels in the rat heart during the late

stage of sepsis, remains to be investigated.

It is of interest to note that lack of change in G

␤

subunit of

GTP-binding protein in the rat heart during sepsis is strikingly

similar to that reported in the liver (22). Furthermore, G

␤

as well

as G

␣

s are found to be quite stable under other pathological

conditions (7, 12, 22). Because chronic infusion of isoprenaline in

rats leads to increases in G

␣

i-2 and G

␣

i-3 mRNA levels whereas

G

␣

s and G

␤

mRNA levels remain unchanged (7), these seem to

suggest that G

␤

and G

␣

s subunits constitute the major

compo-nents of the GTP-binding protein associated signal

transduc-tion pathway that is irresponsive to the external stimuli.

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