INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

AN ALTERATION IN HEPATIC glucose metabolism is one of the key metabolic hallmarks during the progression of sepsis and septic shock. The altered glucose homeostasis is characterized by a rapid depletion of hepatic glycogen content, an impaired glycogenesis, an accelerated glycogenolysis, and a depressed gluconeogenesis (4, 10). The ultimate result of these metabolic alterations is the development of hyperglycemia during the initial stage and a subsequent transition from hyper- to hypoglycemia during the late stage of sepsis (4, 10).

Regulation of liver glucose metabolism is a complicated process that includes many hormonal regulatory factors, such as catecholamines [₁-adrenergic receptor (₁-AR) and ₂-adrenergic receptor (₂-AR) agonists], glucagon, vasopressin, angiotensin, and insulin. ₂-AR agonists and glucagon that interact with plasma receptors lead to the activation of cAMP-dependent protein kinase, which then catalyze the phosphorylation of a number of protein substrates and result in the stimulation of gluconeogenesis, glycogenolysis, and inhibition of glycolysis (19, 34). ₁-AR agonists, vasopressin, and angiotensin that act via changes in intracellular Ca²⁺/calmodulin-linked protein kinases lead to similar changes in gluconeogenic and glycolytic flux as ₂-AR agonists and glucagon. Insulin, in contrast, opposes the action of the above hormones to phosphorylate various protein substrates (19, 34). Catecholamines, as both ₁-AR and ₂-AR agonists, are the major factors that control the homeostatic level of glucose via a dual mechanism involving ₁-AR and ₂-AR mediation (20, 26). Catecholamine-stimulated glycogenolysis occurs primarily through ₂-AR mediation in a variety of species other than rat, including guinea pig, rabbit, cat, and dog (23, 40). Even in the rat liver, a shift from ₁-AR to ₂-AR responsiveness is demonstrable under various conditions (23). Studies with human subjects have indicated that human liver contains ₁-AR and ₂-AR in almost equal proportions (5, 24) and that glucose production via a ₂-AR mechanism is as important as that of ₁-AR on stimulation by epinephrine (35). Previous work from this laboratory has demonstrated that ₂-AR was progressively underexpressed during the early and the late phases of sepsis (42), whereas the -AR was overexpressed during the early but underexpressed during the late stage of sepsis (21). The dynamic changes of both -AR and ₂-AR are likely to serve as a mechanism that contributes to the transition from hyper- to hypoglycemia, especially during the late stage of sepsis.

Recent advances on the molecular pathogenesis of altered receptor function have indicated that transcriptional regulation of receptor mRNA plays a pivotal role in the altered expression of -AR. In heart failure, the density of ₁-AR was reduced and the reduction in ₁-AR density was accompanied by a decrease in the steady-state level of ₁-AR mRNA (6, 18, 33). In cardiac hypertrophy, there was a parallel decrease in ₁-AR density and ₁-AR mRNA level (31). In developing liver, ₂-AR density was decreased, and the decrease in ₂-AR density occurred concomitantly with a decrease in the steady-state level of ₂-AR mRNA (37). To further characterize whether the underexpression of ₂-AR in the rat liver during the progression of sepsis (42) is regulated via transcriptional modification of ₂-AR gene, the present study dealing with transcriptional regulation of ₂-AR gene was undertaken in an attempt to understand the molecular pathogenesis of altered hepatic glucose metabolism during the progression of sepsis.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Animal model. All animal experiments in this study were performed with the approval of the Animal Care Committee of St. 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 to 320 g were used. All animals were fasted overnight with free access to water. They were divided into three groups: control, early sepsis, and late sepsis. Sepsis was induced by cecal ligation and puncture (CLP) by the method of Wichterman et al. (43) with minor modification as described by us (15, 42). Early and late sepsis refers to those animals killed at 9 and 18 h, respectively, after CLP. Preliminary experiments indicate that in septic rats, plasma glucose concentration was elevated by 21% (P < 0.01) during the early phase, whereas it was decreased by 69% (P < 0.01) during the late phase of sepsis (7.0 ± 0.06, 8.5 ± 0.15, and 2.2 ± 0.15 mM for control, early sepsis, and late sepsis, respectively).

Preparation of rat liver crude membranes and [³H]dihydroalprenolol binding to liver membranes. Rat liver crude membranes were prepared according to a procedure described in our previous reports (21, 42). ₂-AR binding assay was carried out using ()-[³H]dihydroalprenolol ([³H]DHA) as a radioligand according to a procedure previously described by us (27, 41, 42) but with modification. The standard assay mixture in a final volume of 0.5 ml contained 10 mM MgCl₂, 50 mM Tris · HCl (pH 7.4), 20 nM [³H]DHA (96 Ci/mmol), and 0 or 20 µM unlabeled ICI-118,551 (a selective ₂-AR antagonist). The specific binding for ₂-AR was defined as the bound radioactivity displaceable by 20 µM of unlabeled ICI-118,551.

Determination of ₂-AR protein level by Western blot analysis. Western blot analysis was performed according to the method of Ausubel et al. (2) with minor modification (15). A rabbit polyclonal antibody (1:500 dilution) raised against a peptide corresponding to amino acids 399-418 mapping at the carboxyl terminus of the ₂-AR of mouse origin (Santa Cruz Biotechnology) was used as a primary antibody. An anti-rabbit Ig, horseradish peroxidase-linked whole antibody (1:1,000 dilution) (Amersham Life Science) was used as a secondary antibody.

Determination of the steady-state level of ₂-AR mRNA by RT-PCR and Southern blot analysis. The steady-state level of ₂-AR mRNA was determined by RT-PCR based on the method of Kawasaki et al. (25) using total cellular RNA extracted from control and septic rat livers. The sense and antisense primers used were designed by the method of Lowe et al. (28) and synthesized by Bio-Synthesis (Lewisville, TX) on the basis of the published cDNA sequences corresponding to ₂-AR (7). The sense primer used was 5'-ACC TCC TTC TTG CCT ATC CA-3', and the antisense primer was 5'-TAG GTT TTC GAA GAA GAC CG-3'. The first strand cDNA was synthesized by using murine leukemia virus reverse transcriptase (Perkin-Elmer). The reaction mixture (20 µl) contained a GeneAmp PCR buffer (50 mM KCl, 10 mM Tris · HCl, pH 8.3, 1.5 mM MgCl₂, and 0.001% gelatin) (Perkin Elmer), 5 mM dithiothreitol, 0.5 µg sample RNA, 1 U of RNasin ribonuclease inhibitor, 50 pmol of oligo d(T)₁₆ primer, 1 mM (each) deoxynucleotide triphosphates (dATP, dCTP, dGTP, and dTTP), and 200 U of reverse transcriptase. The reaction mixture was incubated at 42°C for 30 min, heated to 95°C for 10 min, and immediately cooled to 4°C. The reaction mixture was then diluted with 30 µl of GeneAmp PCR buffer containing 50 pmol of sense primer, 50 pmol of antisense primer, and 1.25 U of Perkin-Elmer AmpliTag DNA polymerase. Cycling conditions for the amplification reaction were a single 5-min heating step at 95°C, followed by 30 cycles of denaturation at 95°C for 20 s, annealing at 54°C for 30 s, and extension at 72°C for 30 s. After the last cycle, the reaction mixture was incubated at 72°C for 7 min and cooled to 4°C. The amplification resulted in an expected product of 559 bp. To ensure that the isolated RNA used for reverse transcription was not contaminated by genomic DNA, an aliquot of each RNA preparation was digested with RNase A (1 U), and the absence of an amplified DNA product after the RNase A digestion was confirmed for each sample. Initially, the number of cycles needed for sufficient but still exponential amplification was titrated. Aliquots of 15 µl were removed from the PCR mixture after different cycles of amplification and were electrophoresed on a 2% agarose gel containing ethidium bromide in 40 mM Tris-acetate, pH 8.6, 2 mM EDTA (1× TAE buffer). Amplified DNA bands were scanned and the relative density was quantified as described earlier. The PCR-amplified product was then used for Southern blot analysis. For internal standard, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was simultaneously amplified using 5'-TGA AGG TCG GAG TCA ACG GAT TTG GT-3' as a sense primer and 5'-CAT GTG GGC CAT GAG GTC CAC CAC-3' as an antisense primer. The amplified PCR fragment for GAPDH was 983 bp.

For Southern blot analysis, an aliquot (40 µl) of PCR-amplified product was separated and transferred onto Magnacharge nylon membranes (Micron Separations) by the method of Sambrook et al. (39). The membranes were prehybridized for 30 min at 42°C in a prehybridization buffer (ECL 3'-oligolabeling system; Amersham Life Science). The prehybridization solution was replaced with a fresh solution containing an oligonucleotide probe specific for ₂-AR (5'-AAG GCA ATC CTG AAA TCT GGA C-3') or GAPDH (5'-CAC GGA AGG CCA TGC CAG TGA GCT TCC CGT-3') labeled with ECL 3'-oligolabeling and detection systems (Amersham Life Science), and the mixture was hybridized overnight at 42°C. After stringent washing, the blots were incubated with a 1:1,000 dilution of an antifluorescein antibody conjugated to horseradish peroxidase (Amersham Life Science) for 1 h at room temperature. The blots were washed and exposed to Hyperfilm-ECL for 40 min. Autoradiographs were scanned, and the relative densities were quantified as described earlier. The mRNA levels were normalized relative to the level of GAPDH to correct for potential difference in the amount of RNA used and the DNA amplified.

Measurement of the transcription rate of ₂-AR mRNA by nuclear run-off assay. Liver nuclei were isolated by the method of Bush (8). Nuclear run-off assay was performed according to the methods of Ausubel et al. (1) and Baeyens and Cornett (3), except that 0.2 µM of FluoroGreen (fluorescein-11-UTP) was used instead of [³²P]UTP. The fluorescein-labeled RNA was hybridized in 1.4 ml of TES-NaCl solution (10 mM Tris · HCl, pH 7.4, 10 mM EDTA, 0.2% SDS, and 300 mM NaCl) with synthetic ₂-AR and GAPDH antisense oligonucleotide probes immobilized on nitrocellulose membranes at 65°C for 48 h. After hybridization the membranes were washed and then incubated with ribonuclease A (10 µg/ml) at 37°C for 30 min. After stringent washing, the blots were incubated with a 1:1,000 dilution of an antifluorescein antibody conjugated to horseradish peroxidase (Amersham Life Science) for 1 h at room temperature. Autoradiographs were scanned, and the relative densities were quantified as described earlier. The transcription rate of ₂-AR mRNA was calculated from the fluorescein-labeled RNA bound to the oligonucleotide probe specific for ₂-AR and corrected for the amount of input-labeled RNA from GAPDH probe.

Stability (half-life) assay of liver ₂-AR gene transcript. The stability of liver ₂-AR mRNA was measured by actinomycin D pulse-chase method (3, 36), with modification as previously described (15). Liver tissue slices were prepared, incubated with actinomycin D, and total RNA was extracted from each sample and subjected to RT-PCR. An aliquot (45 µl) of PCR-amplified product was dot blotted on a nylon membrane and hybridized with fluorescein-labeled synthetic ₂-AR oligonucleotide probe as described for Southern blot analysis. Stringent posthybridization washing of the membranes and detection of the hybridized signals were performed as described earlier. The half-life of ₂-AR mRNA was defined as the time required for the 50% reduction in mRNA level.

Protein assay and statistical analysis. The protein content of hepatic membranes was measured as described by Lowry et al. (29). The statistical analysis of the data was performed using one-way ANOVA followed by Student-Newman-Kuels tests. A P value of <0.05 was considered statistically significant.

Materials. ICI-118,551 HCl [(±)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol] was purchased from Tocris Cookson. ()-[4,6-Propyl-³H]dihydroalprenolol (96 Ci/mmol), FluoroGreen (fluorescein-11-dUTP), ECL Western blotting detection reagent, Hyperflim-ECL, antifluorescein antibody conjugated to horseradish peroxidase, and anti-rabbit Ig, horseradish peroxidase-linked whole antibody were obtained from Amersham Life Science. Rabbit polyclonal antibody raised against a peptide corresponding to amino acids 339-418 mapping at the carboxyl terminus of the ₂-AR of mouse origin was a product of Santa Cruz Biotechnology. Actinomycin D and ribonuclease A were purchased from Sigma. RNasin ribonuclease inhibitor was supplied by Promega. Other chemicals and reagents were of analytic grade.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Because the ₂-AR gene is intronless, it is necessary to confirm that the RNA preparations used for the template of ₂-AR RNA reverse transcription were not contaminated with genomic DNA. Therefore, aliquots of all RNA preparations were digested with RNase A (1 U) before RT-PCR. Figure 1A shows the representative picture of ethidium bromide-stained PCR amplification products with or without RNase A digestion. No amplified DNA products could be revealed in the absence of RNA (lane 1) or after RNase A digestion (lane 2). This confirms the absence of contaminating DNA in the RNA samples used in reverse transcription. The amplified DNA products were increased proportionally with the increasing concentrations of template RNA (lanes 3, 4, 5, and 6 contained 200, 400, 600, and 800 ng of RNA, respectively). Figure 1, A-C, shows that the amplified PCR products derived from specific ₂-AR primers were proportional to the amounts of template RNA and were increased proportionally from 22 to 35 cycles. These results indicate that the RT-PCR used in this study is sufficiently sensitive and accurate to detect changes in ₂-AR mRNA levels during sepsis. On the basis of similar titrations, 30 cycles were adopted for measurement of ₂-AR mRNA and 26 cycles for GAPDH mRNA.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   A: representative picture of ethidium bromide stain of an agarose gel for assessment of DNA-free RNA preparation from liver of control rat. To verify that RNA preparations used as templates for reverse transcription were devoid of contaminating genomic DNA, aliquots of RNA samples were digested with 1 U of RNase A before RT-PCR. +, Presence; , absence of RNase (1 U). B and C: assessment of proportional range of DNA amplification. To define the proportional range of DNA amplification. RT-PCR was performed starting with 200 ng RNA using 22-35 amplification cycles. After each cycle, 15-µl aliquots were taken from reaction mixture and separated on 2% agarose gels with 0.1% of ethidium bromide. Gel was laser scanned, and relative densities of DNA product were quantified by a Jandel Scientific program. B: ethidium bromide stain of an representative agarose gel after different cycles of amplification. C: laser densitometric analysis of cycle-dependent amplification of DNA product using data obtained from Fig. 1B. 2-AR, 2-adrenergic receptor.

Figure 2 depicts changes in the density of ₂-AR in the rat liver during the early and the late phases of sepsis based on [³H]DHA binding studies. The maximal binding capacity (B_max) for [³H]DHA binding was decreased by 12 (P < 0.05) and 35% (P < 0.01) during the early and the late phases of sepsis, respectively. The affinity (the reciprocal of dissociation constant) for [³H]DHA binding was not affected during early and late phases of sepsis (results not shown). These data indicate that the density of ₂-AR in the rat liver was decreased progressively during the course of sepsis. It should be noted that there was a fairly large difference in the B_max number reported in this work versus that reported previously (42) because of the use of different membrane preparations. In the current study, we used homogenate preparation instead of partially purified plasma membrane/light vesicle for better correlation with Western blot analysis.

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Changes in density of 2-AR in rat liver during different phases of sepsis. [3H]dihydroalprenolol ([3H]DHA) binding was performed as described in METHODS. Early sepsis (ES) and late sepsis (LS) refer to measurements performed at 9 and 18 h, respectively, after cecal ligation and puncture. Vertical bars indicate SE. Number of experiments is shown in parentheses within each column.

Figure 3 shows the Western blot analysis of ₂-AR protein level in the rat liver during different phases of sepsis. ₂-AR protein level was decreased by 37 (P < 0.01) and by 72% (P < 0.01) during early sepsis and late sepsis, respectively (Fig. 3B). These findings reinforce the data presented in the previous figure that ₂-AR was underexpressed in the rat liver during the progression of sepsis. It should be mentioned that the magnitude of changes in the ₂-AR density when measured by radioligand assays (decreased by 12 and 35% during early and late sepsis, respectively) and Western blot analysis (decreased by 37 and 72% during early and late sepsis, respectively) may be related to the difference in the specificity of the two methods. Radioligand assays measured total binding of ₁-AR, ₂-AR, and possibly ₃-AR, whereas Western blot analysis detected only ₂-AR because of the use of the ₂-AR-specific antibody.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   A: representative autoradiograph of Western blot analysis. B: Western blot analysis of 2-AR protein level in control and septic rat livers. Western blot analysis was carried out as described in METHODS using rabbit polyclonal antibodies raised against a peptide corresponding to amino acids 399-418 mapping at the carboxyl terminus of the 2-AR of mouse origin. Vertical bars indicate SE. Number of experiments is shown in parentheses within each column. C, control.

Figure 4 shows RT-PCR and Southern blot analysis of the steady-state level of ₂-AR mRNA in the control and septic rat livers. The reverse transcription and amplification of total RNA isolated from liver tissues of control, early septic, and late septic rats resulted in a single band of the expected size of 559 bp using ₂-AR-specific primer and a single band of the expected size of 983 bp using GAPDH-specific primer (Fig. 4A). Analyses of the densitometric signals reveal that the steady-state level of ₂-AR mRNA was decreased by 37 (P < 0.01) and 77% (P < 0.01) during the early and the late phases of sepsis, respectively (Fig. 4B). It should be mentioned that the yields (in mg/g wet wt: 5.4 ± 0.1, 5.5 ± 0.1, 5.5 ± 0.1 for control, early sepsis, and late sepsis, respectively) and the purities (A_260/280: 1.97 ± 0.02, 1.99 ± 0.01, and 1.98 ± 0.04 for control, early sepsis, and late sepsis, respectively) of total RNA remained unaltered among control, early septic, and late septic groups, indicating that changes in the steady-state level of ₂-AR mRNA were not a result of alterations in the isolation procedure for hepatic RNA. These results demonstrate that ₂-AR gene transcript in the rat liver was progressively underexpressed during the course of sepsis.

View larger version (27K): [in this window] [in a new window]   Fig. 4.   A: representative autoradiograph of RT-PCR and Southern blot analysis. B: RT-PCR and Southern blot analysis of steady-state level of 2-AR mRNA in control and septic rat livers. RT-PCR and Southern blot analysis were performed as described in METHODS using total cellular RNA. All 2-AR mRNA levels were normalized relative to level of glyceraldehyde 3-phosphate dehydrogenase (G3PDH) to correct for potential difference in amount of RNA used and DNA amplified.

Figure 5 depicts changes in the transcription rate of ₂-AR mRNA in the control and septic rat livers. Nuclear run-off assays reveal that the transcription rate of the ₂-AR gene was decreased by 36 (P < 0.01) and 64% (P < 0.01) during the early and the late phases of sepsis, respectively (Fig. 5B). It should be mentioned that the yields (in 10⁷ nuclei/g wet tissue: 9.9 ± 0.1, 9.9 ± 0.1, and 9.8 ± 0.2 for control, early sepsis, and late sepsis, respectively) and the viabilities (in %: 95 ± 1, 94 ± 1, and 94 ± 1 for control, early sepsis, and late sepsis, respectively) of hepatic nuclei isolated from control, early septic, and late septic rats were comparable, indicating that changes in the transcription rate of the ₂-AR gene, as showed in Fig. 5B, were not a result of changes in the isolation procedure for hepatic nuclei. These results demonstrate that the rate of synthesis of ₂-AR mRNA was progressively decreased in the rat liver during the course of sepsis.

View larger version (29K): [in this window] [in a new window]   Fig. 5.   A: representative autoradiograph of nuclear run-off assay. B: nuclear run-off assay of the transcription rate of 2-AR mRNA in control and septic rat livers. Nuclear run-off assay was conducted as described in METHODS using specific oligonucleotide probes identical to those of RT-PCR and Southern blot analysis.

Figure 6 shows changes in the stability of ₂-AR mRNA during the early and the late phases of sepsis. The actinomycin D pulse-chase assays reveal that the half-life of ₂-AR mRNA was shortened by 21 (P < 0.01) and 50% (P < 0.01) during early sepsis and late sepsis, respectively (Fig. 6B). These data demonstrate that the rate of degradation of ₂-AR mRNA was increased progressively in the rat liver during the course of sepsis.

View larger version (32K): [in this window] [in a new window]   Fig. 6.   A: representative autoradiograph of half-life assay. B: stability (half-life) assay of 2-AR gene transcript in control and septic rat livers. Half-life of 2-AR gene transcript was measured as described in METHODS using total cellular RNA prepared from liver tissue slices and specific oligonucleotide probes identical to those of RT-PCR and Southern blot analysis. Half-life of 2-AR gene transcript was calculated as time required for 50% reduction in mRNA level.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Recent advances in the studies of molecular biology of ₂-AR have indicated that -AR is encoded by three different genes, ₁, ₂, and ₃, with ₂ being the predominant gene expressed in the liver (20). Using ₂-AR-specific primers for RT-PCR and Southern blot analysis, we found that the steady-state level of ₂-AR mRNA was progressively decreased during the early and the late phases of sepsis (Fig. 4). Furthermore, the decreases in the steady-state level of ₂-AR mRNA during the progression of sepsis were correlated with the declines in the density (Fig. 2) and the protein concentration (Fig. 3) of ₂-AR. These data suggest that changes in the density and the protein level of ₂-AR were a result of the altered expression of ₂-AR gene transcript.

The steady-state level of mRNA measured by RT-PCR and Southern blot analysis is dependent on the transcriptional synthesis of mRNA and its posttranscriptional degradation. In the nuclear run-off assay, the isolated nuclei elongate (but not initiate) RNA transcripts and the incorporation of an RNA precursor is proportional to the transcriptional activity of each specific gene at the time the nuclei are isolated (1). Our findings that the rate of transcription of the ₂-AR gene was decreased during both the early and the late phases of sepsis (Fig. 5) indicate that the reduced rate of synthesis of mRNA encoding ₂-AR was, in part, responsible for the decrease in the steady-state level of ₂-AR mRNA. In the stability assay, the half-life of ₂-AR mRNA was found to be shortened progressively during the early and the late phases of sepsis (Fig. 6), indicating that the enhanced rate of degradation of mRNA encoding ₂-AR was an additional factor contributing to the decrease in the steady-state level of ₂-AR mRNA. On the basis of these findings, it is concluded that the underexpression of ₂-AR gene transcript in the rat liver during the progression of sepsis was regulated by the transcriptional as well as the posttranscriptional mechanisms.

The physiological significance of the altered ₂-AR gene transcript and protein expression in relation to the biphasic changes of glucose metabolism during the progression of sepsis has not been fully explored. Hepatic gluconeogenesis/glycolysis is known to be regulated mainly by catecholamines through ₂-AR and -AR mediations (19, 26, 34). ₂-AR agonist-mediated increases in cAMP level lead to activation of cAMP-dependent protein kinases, which in turn stimulate gluconeogenesis and inhibit glycolysis through phosphorylation of a number of protein substrates, resulting in an increased production of glucose from the liver (34). -AR agonist-mediated increases in intracellular Ca²⁺ and diacylglycerol lead to activation of various protein kinases, including protein kinase C and protein kinase M. These protein kinases generally lead to similar changes in glyconeogenesis and glycolytic flux through catalyzing the phosphorylation of protein substrates. Thus activation of both ₂-AR and -AR elicits a hyperglycemic response (34). The decrease in ₂-AR mRNA expression and the decreased transcription rate and mRNA stability as observed in this study, along with the decrease in ₁-AR density and protein content as reported previously (16, 21), are most likely contributing factors to the formation of hypoglycemia during the late phase of sepsis. During the early phase of sepsis, however, the contribution of the altered ₂-AR density to the formation of hyperglycemia may be limited. Because the receptor binding function and protein expression of ₂-AR are minimally affected during early sepsis, the functional deficit of ₂-AR is likely overridden by the overexpression of ₁-AR gene (16), thus resulting in the production of hyperglycemia. It is also possible that other hormonal effects along with ₂-AR and ₁-AR are involved during the early phase of sepsis.

The mechanism responsible for the decrease in the rate of transcription of ₂-AR mRNA in the rat liver during the progression of sepsis is not clearly understood. Gene synthesis is regulated by pretranscriptional and transcriptional events (30, 38). Pretranscriptional events include signal transduction, second messenger activation, and the activation of transcriptional factors specific to the regulatory region of each specific gene. Transcriptional events include the initiation of RNA synthesis, elongation of the nascent RNA chain, and termination of RNA synthesis. ₂-AR gene in rat liver contains no introns and has two transcripts, a major 2.2-kb and a minor 1.6-kb species (22). Primer extension and RNase protection analyses of rat ₂-AR gene have identified two transcription start points (tsp) at 64 (tsp 1) and 220 (tsp 2), the latter of which is analogous to the single tsp identified at the same location in human ₂-AR gene (17). Fragments 36 to 100 (promoter 1; P1) and 186 to 312 (promoter 2; P2) are sufficient to promote transcription, whereas fragment 911 to 1122 contains a negative regulatory element (22). In addition, two DNA elements, cAMP response element (CRE), and glucocorticoid response element, within the 5'-flanking region may participate in regulating the transcription of ₂-AR gene (12, 13). Cell-specific control of gene transcription requires the availability of a correct set of DNA-binding proteins as transcription factors that associate with DNA response elements (9). A variety of transcription factors, such as CCAAT/enhancer binding protein (C/EBP) and CRE binding protein (CREB), have been reported to affect the transcription of hepatic ₂-AR gene (12, 22). C/EBP-, a member of the C/EBP family of basic leucine zipper transcription factors, has been proposed to function as a negative regulator for hepatic ₂-AR gene expression in vivo because a decrease in C/EBP- level was found to correlate with an increase in ₂-AR mRNA level in the remnant liver after partial hepatectomy (22). Because C/EBP- level has been reported to be increased in the rat liver during the progression of sepsis (11), it is conceivable that an increase in the level of C/EBP- is responsible for the sepsis-induced decrease in the transcription of ₂-AR gene transcript, as reported in the current study. CREB, a transcription factor of 43 kDa capable of stimulating target gene transcription through phosphorylation, has been reported to play a positive feedback role on ₂-AR gene expression (12), because a decrease in CREB level was found to correlate with a decrease in ₂-AR mRNA level in the lung and DDT1-MF2 cells after prolonged agonist stimulation (32). Because circulating catecholamines were increased after the onset of sepsis and their levels remained elevated throughout the progression of sepsis (21, 41), it is possible that CREB level may be decreased in response to the elevated circulating catecholamines and that the decrease in the CREB level, in turn, leads to the decrease in the hepatic ₂-AR gene expression, as observed in the current study. Further investigation of the possible participation of various transcription factors may shed light on the exact mechanism leading to the underexpression of ₂-AR gene transcript in the liver during the progression of sepsis.

It has been reported that the age-related reduction in the abundance of ₂-AR mRNA was associated with a destabilization of its gene transcript in the liver (3). In other cell types such as DDT1-MF2 cells, an agonist-promoted reduction in the ₂-AR mRNA abundance was reported to correlate with a decrease in the half-life of ₂-AR mRNA (14). There are a number of factors known to influence the stability of the ₂-AR gene transcript. These include changes in the length of the transcript poly(A)⁺ tail, changes in the property of poly(A)⁺ binding proteins, and the presence of either stem-loop structures or AU-rich motifs in the 3'-untranslated region (3, 13). Many unstable mRNAs have one or more copies of an AU-rich motif related to (AUUU)n or (AUUUA)n in their 3'-untranslated regions (3). It is of interest to note that rat ₂-AR mRNA has four AUUU motifs in the 3'-untranslated region that are capable of regulating its transcript stability (3, 17). Further studies are required to elucidate the underlying mechanism leading to the instability of ₂-AR gene transcript in the rat liver during the progression of sepsis.

Perspectives