INTRODUCTION

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SEVERAL REGULATORY SYSTEMS function to adjust the disturbance in resting homeostasis due to exercise stimulus. The peripheral sympathoadrenal system exerts a major integrative function in homeostasis. Both as a neurotransmitter and as a hormone, catecholamines (CAs) exert control over several physiological and metabolic functions, resulting in the ability to adjust to the stress of physical exercise (13).

Tyrosine hydroxylase (TH) catalyzes the hydroxylation of tyrosine, producing dopamine, which is the rate-limiting step in CA biosynthesis (18). Dopamine--hydroxylase (DH) is another important CA biosynthetic enzyme that catalyzes the conversion of dopamine to norepinephrine. The increase in CA biosynthesis in response to a variety of factors is associated with the elevated activity of CA biosynthetic enzymes (13, 24). TH enzyme activity is particularly important, and it is time dependently controlled by different mechanisms (9). Short-term (rapid) regulation of TH activity is mediated posttranslationally, whereas long-term (prolonged) regulation of the enzyme occurs at the genomic level, leading to changes in TH gene expression and TH mRNA levels. Many, but not all, factors regulating the expression of TH also regulate the expression of DH (14, 15). Like many of the other stress-responsive genes, the genes encoding TH and DH contain activator protein (AP)-1-like binding sites in their promoters (TH reviewed in Refs. 9, 24, 25; DH reviewed in Refs. 14, 25, 27). AP-1 factors could be important in establishing stress-induced patterns of gene expression in different tissues (16). AP-1 is a dimer composed of various combinations of Fos- and Jun-like proteins, and AP-1 complexes interact with AP-1 sites present in the promoter regions of target genes (16). The induction of c-fos is typically transient, whereas long-lasting Fos-related antigens (FRAs), including FRA-2 and fosB, are elevated after repeated exposure to stressful stimuli (8, 19). The induction of FRA-2 and increased AP-1-like binding activity is associated with the sustained transcriptional activation of TH and DH genes in the rat adrenal medulla (19).

In addition to CAs, neuropeptide Y (NPY) is synthesized in the adrenal medulla and is coreleased with epinephrine and norepinephrine (12). Often, the regulation of NPY biosynthesis parallels that of CA biosynthesis (6, 28). NPY may also play an important role in the autocrine regulation of TH gene expression and activity (7, 8). Hiremagalur et al. (6) proposed that NPY may be part of the homeostatic mechanisms that are involved in the adaptation to stress. Submaximal endurance training (SET) increases the ability to synthesize not only CAs but also NPY under conditions of stress (11).

It is known that varying durations, repetitions, intensities, and types of stress elicit different responses on the phases of transcriptional activation of the adrenal medullary CA biosynthetic enzymes (25). For example, a single exposure to cold or immobilization stress causes a rapid and temporary increase in TH mRNA levels; however, repetition of the same stressors causes a persistent activation of the genes encoding TH and DH in the adrenals (25, 29). This discrepancy is most likely due to the activation of different transcriptional pathways, i.e., whereas c-fos is induced primarily by a single stressful stimulus, repeated immobilization leads to the induction of other FRAs in the adrenal medulla (19). However, an increase in TH activity and protein level can be seen only after repeated stress.

We tested the hypothesis that SET elevates the expression of CA biosynthetic enzymes TH and DH mRNAs and examined the relationship of these changes to the AP-1 regulatory element and FRAs. In addition, we also hypothesized that SET elevates NPY mRNA expression. We determined TH activity, TH protein level, TH, DH, and NPY mRNA levels, AP-1 binding activity, and the immunoreactivity of FRAs, including FRA-2 in the adrenal medullae of exercise-trained and control rats.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Animals. This experimental protocol was approved by the University of Florida Animal Care and Use Committee and followed the guidelines established by the American Physiological Society for the use of animals in research. Twenty adult (7 mo old), female Sprague-Dawley (Harlan Sprague-Dawley, Indianapolis, IN) rats were randomly assigned to either a sedentary control group (n = 10) or an exercise training group (n = 10). Rats were housed individually and maintained on Purina Rat Chow and water ad libitum with a 12:12-h light-dark cycle (0600 to 1800). Exercise training sessions were begun 60-90 min after the beginning of the dark cycle. Training sessions were performed during the animals' nocturnal (dark) cycle by using a dim red light for illumination. Animals were housed and trained in the same room at 22 ± 2°C. Animals were weighed at the beginning and at the end of the study.

Exercise training protocol. Animals were exercised (90 min/day, 5 days/wk) for 12 wk on a motor-driven treadmill, as detailed previously (21, 32). Both the treadmill speed and grade were gradually increased over the course of the 12-wk training period to provide a training overload throughout the training regimen up to 30 m/min at 18% grade for 90 min. The treadmill grade and speed required to approximate a relative workload throughout the training protocol were estimated from oxygen cost of running studies performed in our laboratory (10). Although a precise estimation of a specific, relative workload in exercising rats is difficult, our training intensity was designed to elicit ~70-75% maximal oxygen uptake and was based on previous work using treadmill running in adult rats (10). Regardless of the exact relative workload during the training program, it is important to note that this specific training protocol results in significant improvements in both oxidative and antioxidant enzyme activity in adult rat skeletal muscles and diaphragm (4, 21, 32). Thus we are confident that our training regiment resulted in significant biological adaptations in our animals.

Tissue preparation. Animals were anesthetized with pentobarbital (90 mg/kg ip); adrenal glands were removed quickly and immediately frozen by immersion in liquid nitrogen. Tissues were stored at 80°C. At the time of the assay, adrenal glands were decapsulated and the medullae were separated from the cortex. Adrenal medullary preparations were weighed and homogenized in 100 µl of phosphate buffer (2 mM NaPO₄, 0.2% Triton, pH 7.0). Protein amount was determined by the method of Bradford (3).

TH activity. TH activity was measured using a radioenzymatic assay as described previously (28) that is based on a modification of the assay by Reinhard and colleagues (23) using cofactor (6-methyl-5,6,7,8-tetrahydropterin HCl, 1.5 mM) and [3,5-³H]tyrosine (100 µM; 1 µCi/reaction).

TH immunoreactivity. TH protein levels were determined by using our previously described methods (28) using polyclonal antibody to TH IgG (Pel-Freez Biologicals, Rogers, AR) and horseradish peroxidase-labeled donkey anti-rabbit IgG (Amersham Life Sciences, Arlington Heights, IL).

TH, DH, and NPY mRNA levels. TH, DH, and NPY mRNA levels were determined in the adrenal medullae by dot blot analysis with ³²P random primer-generated probes, as previously described (29). Nylon membranes were stripped and rehybridized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Specific probes were determined by Northern analysis.

Preparation of nuclear protein extracts. Adrenal medullae from five rats were pooled and homogenized by using our laboratory's previously published method (29). Protein amount was determined by the method of Bradford (3).

Electrophoretic mobility shift assay. Electrophoretic mobility shift assay used in the present study was based on our laboratory's previously published method (29). The AP-1 25-base oligonucleotide from Promega (Madison, WI) was ³²P 3' end-labeled with the End Labeling System (Promega).

Immunoblots for FRAs analysis. FRAs and FRA-2 in the adrenal medulla were analyzed by immunoblots as previously described (19). Frozen adrenal medullae from individual animals were homogenized on ice in 20 mM HEPES, pH 7.5, 350 mM NaCl, 25% glycerol, 0.25% Nonidet P-40, 1 mM Na₂CO₃, 0.25 mM phenylmethylsulfonyl fluoride, 5 mM MgCl₂, and 1 µg/ml each of aprotenin, pepstatin, and leupeptin, 1 mM EGTA, and 1 mM DTT. The homogenates were subsequently clarified by centrifugation. Protein concentration was determined by using the Bradford assay. Equal amounts of proteins were separated on 10% SDS-PAGE and electroblotted onto a nitrocellulose membrane (Bio-Rad). The membranes were blocked by incubation with 5% nonfat milk in TBST (50 mM Tris, pH 7.5, 500 mM NaCl, 0.05% Tween). The membranes were incubated with the primary antibody (1:4,000 in TBST, 5% BSA) at 4°C overnight. An antibody with broad specificity for FRAs (kindly provided by Dr. M. Iadarola, National Institutes of Health, Bethesda, MD) or antisera to FRA-2 (rabbit polyclonal antibody, SC-604, from Santa Cruz raised against a peptide corresponding to amino acids 3-22 mapping at the amino terminus of FRA-2 of human origin) were used in these experiments.

Statistical analysis. Data are expressed as means ± SE. Means were compared by Student's t-test (two-tailed, unpaired). A probability value of <0.05 was considered significant.

	RESULTS

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We examined the effects of SET on TH activity, TH immunoreactivity, TH mRNA, DH mRNA, and NPY mRNA levels in the adrenal medulla. Additionally, AP-1 transcription factor binding activity and FRA-2 and late FRAs immunoreactivity were also examined in the tissues from the same animals.

Body weights and adrenal weights. Both control and exercise-trained animals showed a similar increase in body weight during the 12-wk experimental period, indicating that SET did not affect weight gain throughout the study. The initial and final body weights were 257 ± 5 and 296 ± 5 g, respectively (control group), and 259 ± 6 and 283 ± 7 g, respectively (SET group).

TH activity and TH protein levels. Total protein amounts in the adrenals were 2.81 ± 0.12 mg (control group) and 2.87 ± 0.08 mg (SET group). TH activity was significantly elevated by 62% in the exercise-trained animals compared with the sedentary controls (98 ± 6 vs. 59 ± 4 nM · mg protein¹ · h¹; P < 0.05).

View larger version (40K): [in this window] [in a new window]   Fig. 1.   Effect of submaximal endurance training on tyrosine hydroxylase (TH) immunoreactivity. Representative Western blots of control (C) and trained (T) rats are shown. Bands correspond to 39 kDa.

TH, DH, and NPY mRNA levels. The effect of SET on the expression of several mRNAs was also assessed. mRNA levels of TH in the exercise-trained rats were 40% greater than those of the controls (P < 0.05; Fig. 2). Similarly, mRNA levels of DH were significantly elevated by 70% in the exercise-trained group (P < 0.05; Fig. 2). We also investigated the effect of SET on NPY mRNA levels in the adrenal medulla. Similarly, there was an 80% exercise-induced elevation in NPY mRNA (P < 0.05; Fig. 2). There was not any significant difference in GAPDH mRNA levels between the control and exercise-trained groups.

View larger version (32K): [in this window] [in a new window]   Fig. 2.   Effect of submaximal endurance training on TH, dopamine--hydroxylase (DH), and neuropeptide Y (NPY) mRNA levels in rat adrenal medullae. Data are means ± SE of 10 rats. * Significantly different from respective controls (P < 0.05). Inset: representative dot blots. OD, optical density.

AP-1 transcription factor binding activity. The enhanced enzyme expression and the synthesis with SET suggest an elevated activity of CA biosynthetic pathway. Therefore, we examined whether the exercise-induced elevation in TH mRNA is associated with an exercise-induced increase in AP-1 transcription factor binding activity. The adrenal medullae from five rats in each group were combined to assess the level of AP-1 transcription factor binding to DNA as determined from gel shift assays. The amount of nuclear protein recovered from the adrenal medulla was comparable in each group (data not shown). The specificity of transcription factor binding was verified by competition with excess, unlabeled AP-1 consensus oligonucleotide or excess, unlabeled oligonucleotide corresponding to the AP-2 binding site. Unlabeled AP-1 consensus oligonucleotide competed with labeled AP-1 oligonucleotide, whereas unlabeled AP-2 oligonucleotide did not. The binding activity of AP-1 in the adrenal medulla was 78 ± 3% higher in exercise-trained animals (Fig. 3).

View larger version (91K): [in this window] [in a new window]   Fig. 3.   Effect of submaximal endurance training on activator protein (AP)-1 binding in rat adrenal medullae. AP-1 oligonucleotide was incubated with nuclear extracts from control animals (lanes 4 and 5) and trained animals (lanes 6 and 7). Adrenal medullae from 5 rats were pooled to prepare each nuclear protein extract. Lane 1 represents labeled AP-1 oligonucleotide only; lane 2 represents labeled oligonucleotide and nuclear protein extract with unlabeled AP-1 consensus sequence (competitor); lane 3 represents labeled oligonucleotide and nuclear protein extract with unlabeled AP-2 consensus sequence (noncompetitor).

FRA-2 immunoreactivity and late FRAs. FRA-immunoreactive proteins are induced in many different brain regions after different repeated or chronic treatments. However, our findings from the adrenal medullae of the rats subjected to SET demonstrated that immunoreactive FRAs, including FRA-2, were not elevated by this exercise protocol (Fig. 4). In contrast, we observed a tendency to decrease in FRA-2 immunoreactivity and the late FRA immunoreactivity (39.2 ± 10.2 vs. 54.1 ± 15.0 arbitrary units and 342.2 ± 50.4 vs. 505.5 ± 145.3 arbitrary units, respectively) in the exercise-trained animals, but these decreases were not significant.

View larger version (11K): [in this window] [in a new window]   Fig. 4.   Effect of submaximal endurance training on the levels of Fos-related antigen (FRA)-2 (top) and FRA family immunoreactive proteins (bottom) in rat adrenal medulla. Representative Western blot analysis of total protein homogenate from individual animals. Five micrograms of protein were loaded for each sample. Mobility of the marker protein is indicated.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The primary finding of the present study is that SET performed at an estimated intensity of 70-75% of maximal oxygen uptake is associated with increased levels of TH activity, TH protein, TH, DH, and NPY mRNA expressions in the adrenal medullae of adult, female Sprague-Dawley rats. Moreover, the results provide evidence that the exercise-induced increases in TH, DH, and NPY mRNA expressions are associated with the increase in AP-1 binding activity but not in the FRAs, including FRA-2.

The increase in CA biosynthesis that occurs in response to repeated or chronic exposure to stressors is associated with elevated activity of the CA biosynthetic enzymes, and these changes reflect increases in the amount of the enzyme protein (25). It is well established that TH enzyme activity is regulated by different mechanisms (1) and that it increases with the enhanced synthesis and release of CAs (33). A short-term increase in the demand for CA release usually leads to enhanced phosphorylation of TH (17); however, a more prolonged need for increased TH activity results in enhanced levels of TH mRNA, probably due to an augmentation of TH gene transcription (2). Although we did not measure plasma CA levels in the present study, we demonstrated the SET-induced increase in the activity, protein amount, and mRNA levels of TH, and in the mRNA level of DH, indicating that SET induces an increased activity in the CA biosynthesis pathway. These results are in contrast with our laboratory's previous report, using low-intensity endurance training, in which TH mRNA levels were reduced (30). The major difference in the exercise-training protocols used in these two studies from our laboratory is the intensity of the exercise, suggesting that the intensity of the endurance training alters the long-term consequences in CA biosynthetic enzymes.

Having demonstrated the stimulating effect of SET on CA biosynthetic enzyme production, we examined whether this change is associated with changes in interactions of certain transcription factors with previously identified functional promoter elements on the genes encoding TH and DH. Several genomic regulatory elements have been identified in the promoter region of the TH gene; two of the most important are AP-1 regulatory element and the cAMP response element. The rapid induction of TH and DH transcription in response to short-term exposure to stress probably reflects posttranslational activation of preexisting factors, including the cAMP response element binding protein (25). However, longer exposures to stress gradually decrease the amount of immunoreactive phosphorylated cAMP response element binding protein (25). On the other hand, we have shown previously that the transcriptional activation of TH in response to long-term cold exposure is correlated with increased binding of AP-1 transcription factor to AP-1 regulatory element (31). Many stress-responsive genes, including not only TH but also DH, contain AP-1-like binding sites in their promoters, and AP-1 factors could be important in establishing stress-induced patterns of gene expression (19). The exercise protocol used in the present study increased the binding activity of AP-1 transcription factor and the levels of TH and DH mRNAs consistently. The AP-1 regulatory element binds the products of the immediate early genes, the c-Fos- and c-Jun-related proteins. AP-1 transcription factor is a dimer of these proteins. However, members of the AP-1 transcription factor family are differentially regulated by single and repeated stress in the rat adrenal medulla, suggesting distinct roles in establishing stress-induced patterns of gene expression in this tissue (19). For example, adrenal TH and DH mRNA levels in response to repeated immobilization stress were not significantly different between c-fos knockout animals and wild type, indicating that c-Fos is either not involved in this response or that it can be compensated for by another factor (26). c-fos expression is typically transient, whereas repeated stress stimuli markedly elevate long-lasting FRAs, including FRA-2 and late FRAs (19). Therefore, we also investigated the possible changes in the FRAs. In contrast to expectations, we did not observe a significant change in their levels. This finding suggests that another immediate early gene product, rather than FRA-2 and FRAs, can be associated with the increase in AP-1 transcription factor binding in response to SET. On the other hand, transcription was not directly measured in the present study; therefore, the changes might also result from either the decreased mRNA turnover or the increased mRNA stability (5, 25). These possibilities remain to be clarified.

In the present study, we also examined the effect of SET on NPY mRNA because there is evidence of a role for NPY in the autocrine regulation of TH mRNA expression and activity (7). NPY mRNA expression often increases concomitantly with TH gene expression. Several stimulators of TH gene expression also increase NPY gene expression in the adrenal medulla (6). For example, we have demonstrated that carbachol, a cholinergic agonist, stimulates both TH and NPY mRNA expression (28). Similarly, it has been shown that long-term physical activity enhances the ability to synthesize both NPY and CAs under conditions of stress (11). In the present study, NPY mRNA expression was also stimulated by SET.

In summary, our data demonstrated that SET increases adrenomedullary TH activity, TH immunoreactivity, and TH mRNA, increases the mRNA levels of DH and of NPY, and the binding activity of AP-1 transcription factor, but not the levels of FRAs.

Finally, these data, coupled with our laboratory's previous report using low-intensity endurance training, suggest that 1) exercise intensity is a pivotal factor in the effect of SET on adrenomedullary CA biosynthesis, 2) another immediate early gene product rather than FRAs and FRA-2 may be involved in the increased AP-1 transcription factor binding activity due to SET, and 3) the correlation between the expressions of TH and NPY mRNAs is maintained in response to SET.