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-adrenergic-receptor mRNA level
measured by competitive RT-PCR
1 Gene Experiment Center, ABSTRACT Fujii, Nobuharu, Takeshi Shibata, Sachiko Homma, Haruo
Ikegami, Kazuo Murakami, and Hitoshi Miyazaki. Exercise-induced changes in
reverse transcription-polymerase chain reaction; upregulation; catecholamine; lymphocyte
INTRODUCTION THE SYMPATHOADRENOMEDULLARY SYSTEM plays an essential
role in regulating physiological function during exercise. Many of its effects are mediated by It would be of great value to know whether exercise-induced
upregulation of The development of the polymerase chain reaction (PCR), a new and
extremely sensitive method of quantifying small amounts of DNA and
mRNA, has made it possible to study the expression of genes in very
small tissue samples (30). The competitive reverse transcription
(RT)-PCR in particular can be used to obtain quantitative data
regarding mRNA levels comparable to traditional RNA blot techniques,
with the added advantages of PCR (12, 32). In this study, we used the
competitive RT-PCR method to investigate the changes in MATERIALS AND METHODS
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
-adrenergic-receptor mRNA level measured by competitive RT-PCR. J. Appl. Physiol. 82(6):
1926-1931, 1997.
Competitive reverse transcription-polymerase
chain reaction (RT-PCR) analysis was used to clarify whether dynamic
exercise-induced increases in -adrenergic-receptor (-AR) number
in human lymphocytes are accompanied by increases in the -AR mRNA
level. Sixteen healthy subjects performed cycle ergometry until
exhaustion. Before and immediately after exercise, peripheral blood was
drawn from a forearm vein for preparation of lymphocytes. Both the
-AR mRNA level and the -AR number were significantly increased by
exercise. The changes in -AR mRNA level and -AR number were
significantly correlated (r = 0.63, P < 0.01). This finding suggests
that a rapid increase in -AR mRNA level might be an early adaptive
response of the sympathetic nervous system to dynamic exercise. In
vitro incubation of lymphocytes with epinephrine had no effect on
-AR mRNA levels, nor did adenosine 3
,5-cyclic
monophosphate, protein kinase C, or intracellular
Ca2+ increase the -AR mRNA
level in vitro. Therefore, it appears that other mechanisms underlie
the exercise-induced elevation of -AR mRNA levels in human
lymphocytes.
-adrenergic receptors (-AR) in the cell membrane. In the basal condition, the information of -AR signal transduction components in human lymphocytes is significantly correlated with that in various tissues (3, 18, 22, 27, 28).
Especially, the number of -AR and the responsiveness of -AR to an
agonist (3, 28), and the content of stimulatory guanine nucleotide
binding protein (18) in human lymphocytes, are significantly correlated
with those in the myocardium. Dynamic exercise induces increases in
-AR number in human lymphocytes (2, 5, 13, 15), and identical
increases in -AR number have been demonstrated in the myocardium of
the rat (19). For this reason, lymphocytes derived from peripheral
blood are often utilized as a model for determining the effects of
acute dynamic exercise on the -AR system.
-AR is accompanied by activation of -AR gene expression. However, this has never been investigated, in part because
of the difficulty of obtaining sufficient numbers of lymphocytes to quantify low levels of -AR gene expression. This unavailability of sufficient samples also made it very difficult in the past to
simultaneously evaluate changes in -AR number and mRNA level because
traditional methods, such as Northern and dot blotting, are incapable
of quantifying -AR mRNA in the small numbers of lymphocytes
remaining after determination of the -AR number by binding assay.
-AR mRNA
level in human lymphocytes after acute dynamic exercise.
-AR number.
Total -AR number in intact lymphocytes was determined by measuring
the binding of the lipophilic radioligand
[125I]iodocyanopindolol
([125I]ICYP) in the
presence and absence of 2 µM propranolol. For the binding assay,
intact lymphocytes (106 cells)
were incubated with various concentrations (1-180 pM) of
[125I]ICYP in 0.5 ml
of buffer A containing 0.5% bovine
serum albumin. The difference in
[125I]ICYP binding
with and without propranolol represents total specific binding to
-AR. Incubation was carried out at 37°C for 40 min and stopped
by placing the tubes on ice and adding 2 ml of ice-cold PBS containing
0.1% bovine serum albumin to each tube. The samples were then filtered
through Whatman GF/D filters, and each filter was washed three times
with an additional 3 ml of PBS. The radioactivity of the filters was
determined in a gamma counter (ARC1000M, Aloka). The binding data were
analyzed according to the method of Scatchard (31).
Isolation of total RNA.
Total cellular RNA was isolated by Isogen (Nippon Gene). Briefly, cells
were homogenized by pipetting in a mixture containing Isogen and
chloroform. The extracted RNA was precipitated with isopropanol and
washed with 70% ethanol. RNA was checked by 1% agarose gel
electrophoresis in the presence of 0.66 M formaldehyde. The purity and
yield of total RNA were determined spectrophotometrically by measuring
the absorbance of aliquots at 260 and 280 nm.
Effect of epinephrine stimulation in vitro.
The effect of epinephrine stimulation on the -AR mRNA level in vitro
was tested on lymphocytes obtained from two subjects. Immediately after
lymphocyte isolation, a portion of the cells (4 × 106 cells) was suspended in RPMI
1640 medium containing superoxide dismutase and catalase (1 mg/ml final
concentration, respectively) to prevent epinephrine oxidation and was
preincubated for 30 min at 37°C. Then, various concentrations of
epinephrine were added (10
10 to
103 M final concentration),
and the cells were incubated for 30 min at 37°C in a total volume
of 1 ml. The reaction was terminated by placing the tubes on ice. After
centrifugation for 5 min at 200 g at
4°C, the supernatant was removed and pellets were immediately submitted for isolation of total RNA. The effects of forskolin (107 M final
concentration), a direct activator of adenylyl cyclase, phorbol
12-myristate 13-acetate (PMA;
107 M final concentration),
a direct activator of protein kinase C (PKC), and A-23187
(107 M final
concentration), a Ca2+ ionophore,
were also tested by the same procedure.
Preparation of a deletion-mutated -AR cDNA fragment
for competitive RT-PCR analysis.
A 401-base pair (bp) fragment containing part of the coding region of
the human 2-AR gene
(73-473; nucleotides are numbered sequentially from the
translation initiation site) was amplified with the following primers:
5-ACGCAGCAAAGGGACGAG-3 (5 sense primer) and
5-CACACCATCAGAATGATCAC-3 (3 antisense primer). These fragments were phosphorylated at their 5 ends and inserted into the EcoR V site of pBluescript KS(+) (Stratagene, La Jolla, CA).
The resultant plasmid was digested with Msc I and BstE II, blunt ended
with T4 DNA polymerase, and self-ligated to generate a plasmid
containing an insert lacking the Msc I-BstE II fragment (62 bp). This
plasmid was amplified by PCR by using the primers described above, and
the resultant deletion-mutated
2-AR cDNA (339 bp) was resolved
by polyacrylamide gel electrophoresis. After recovery, the cDNA
fragment was quantified and used as a competitor for competitive
RT-PCR.
Competitive RT-PCR.
Total RNA (1.8 µg) was reverse transcribed by using random primers. A
corresponding aliquot of cDNA mixture synthesized from 100 ng total RNA
was applied to competitive PCR analysis by using the same primers
described above in the presence of various amounts of the competitors
with a trace amount of
[
-32P]dCTP to
quantify the PCR products. PCR was performed by 28 cycles of
denaturation at 94°C for 1 min, annealing at 56°C for 1 min, and extension at 72°C for 1.5 min. Incubation was then continued at
72°C for another 8.5 min to complete the polymerization. The PCR
products were size fractionated on 5% acrylamide gels. The gels were
dried and analyzed by using computer-based imaging system BAS 2000 (Fuji). The amount of -AR mRNA was then calculated by extrapolating
from the intersection of the curves, where the amounts of target and
competitor were equivalent [log(target/com- petitor) = 0] to the x-axis. Resultant
-AR mRNA values were normalized with the RT-PCR products of
glyceraldehyde-3-phosphate dehydrogenase mRNA quantified in an
independent experiment series (9).
Determination of plasma catecholamines and cortisol concentrations.
Plasma norepinephrine and epinephrine concentrations were quantified by
high-performance liquid chromatography, and plasma cortisol
concentrations were determined by radioimmunoassay.
Statistics.
The significance of differences between values obtained before and
after exercise was assessed by using the two-tailed Student's paired
t-test. The results of in vitro
experiments were compared by analysis of variance. Pearson's formula
was used to define the correlation coefficients. A probability value of
<0.05 was considered significant. All data are expressed as means ± SE.
RESULTS
The subjects exercised for 20.6 ± 1.0 min, and their mean maximal
O2 uptake was 58.5 ± 3.3 ml · kg1 · min1.
Maximal bicycle ergometry significantly increased the number of -ARs
to 98.8 ± 18.5% over resting levels
(P < 0.01; Fig. 1). There were no significant changes in
the affinity of the receptor for
[125I]ICYP, a
lipophilic -AR antagonist.
-adrenergic-receptor (-AR)
number. Values are means ± SE; n = 16. -AR number was determined by Scatchard analysis of
[125I]iodocyanopindolol
binding in presence and absence of 2 µM propranolol.
The validity of competitive RT-PCR for quantifying the -AR mRNA
levels was examined before detailed quantification of the -AR mRNA
level. A cDNA mixture containing -AR mRNA was diluted to 50, 25, and
12.5% and subjected to competitive RT-PCR as shown in Fig.
2. The calculated -AR mRNA levels
correlated well with the input amounts of the cDNA mixtures,
demonstrating the validity of our method. Figure
3, A and
B, shows representative data obtained by competitive RT-PCR analysis. The -AR mRNA level was found to have
increased after exercise when compared with the resting level. As
summarized in Fig. 4, the -AR mRNA level
was significantly elevated by exercise
(P < 0.05). The changes in -AR
mRNA level were significantly correlated with the changes in -AR
number (r = 0.63, P < 0.01; Fig.
5).
-AR mRNA was diluted to
50, 25, and 12.5% and subjected to competitive RT-PCR analysis. One
arbitrary unit represents amount of reverse-transcribed target product
obtained by competitive PCR with a 100% concentration of cDNA
mixture.
-AR mRNA levels.
A: representative findings of
competitive RT-PCR for -AR mRNA in lymphocytes prepared at rest and
after exercise. Amounts of competitor added to cDNA mixtures were 5, 10, 20, 40, 60, and 100 fg, and amplification was performed for 28 cycles. bp, Base pairs. B: analysis of
-AR mRNA levels. Amounts of target cDNA in unknown samples were
calculated from equivalence point [log(T/C) = 0].
-AR mRNA level determined
by competitive RT-PCR analysis. Values are means ± SE; n = 16.
-AR number and changes in -AR
mRNA level. Each circle represents an individual data point in which
-AR number is graphed as a function of -AR mRNA level in that
same sample.
Plasma epinephrine, norepinephrine, and cortisol concentrations at rest
and immediately after exercise are shown in Table 1. The correlation coefficient between the
changes in plasma epinephrine concentration and the changes in -AR
mRNA level was 0.44 but did not reach the level of statistical
significance (P = 0.09). No
correlations were found between the changes in plasma norepinephrine
(r = 0.05, P = 0.87) or cortisol
(r = 0.06, P = 0.83) concentrations and the
changes in -AR mRNA level.
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In in vitro experiments, incubation of lymphocytes with various
concentrations of epinephrine for 30 min did not raise the -AR mRNA
level (Fig. 6), nor did the direct adenylyl
cyclase activator forskolin, the PKC activator PMA, or the
Ca2+ ionophore A-23187 have any
effect on the -AR mRNA level (Fig. 7).
-AR mRNA
level in vitro. Values are means ± SE for 2 independent experiments
performed in triplicate. Various concentrations of adrenaline (final
concentration 1010 to
103 M) were added, and
cells were incubated for 30 min at 37°C.
-AR mRNA level in
vitro. Values are means ± SE for 2 independent experiments performed in triplicate. Lymphocytes were treated with
107 M of forskolin, phorbol
12-myristate 13-acetate (PMA), and A-23187, or
106 M epinephrine for 30 min at 37°C.
DISCUSSION
RT-PCR analysis has been shown to be 1,000- to 10,000-fold more sensitive than traditional RNA blotting techniques (12, 32). However, it is difficult to obtain quantitative information by RT-PCR analysis because of the exponential nature of PCR amplification. In competitive RT-PCR, an internal control containing the same primer template sequences as the target cDNA makes it possible to determine the absolute amount of target cDNA by allowing known amounts of competitor DNA to compete with the target for primer binding during amplification. Thus competitive RT-PCR is effective in quantifying the mRNA level in small samples such as the lymphocytes in blood derived from human subjects.
In this study, we demonstrated that the exercise-induced increase in
-AR number in human lymphocytes is associated with upregulation of
its mRNA level. A significant correlation between the changes in -AR
mRNA level and in -AR number was demonstrated. In general, long-term
exposure of cells to agonists is followed by downregulation of -AR
(i.e., a decrease in -AR number), accompanied by a decrease in
-AR mRNA level (17). In contrast, it has been reported that short-term (30-min) exposure of
DDT1MF-2 hamster smooth muscle cells to 100 nM of epinephrine activates the rate of
2-AR gene transcription,
resulting in an increase in steady-state
2-AR mRNA level (7). This
effect is mediated by elevation of intracellular adenosine
3,5-cyclic monophosphate (cAMP) levels. The human 2-AR promoter region contains
the sequence GTACGTCA, which functions as the cAMP response element and
promotes 2-AR gene
transcription (6). In this study, the plasma epinephrine concentration
increased from 0.15 nM at rest to 0.94 nM after exercise. The change in plasma epinephrine concentration yielded a higher correlation coefficient (r = 0.44) with the change
in -AR mRNA level than the change in plasma norepinephrine
concentration (r = 0.05) but did not
reach the level of statistical significance
(P = 0.09). We therefore examined the
effect of epinephrine stimulation on the -AR mRNA level in isolated
lymphocytes. However, no changes in -AR mRNA level were found, not
only at physiological concentrations, i.e.,
1010 M, the plasma
epinephrine concentration at rest, or
109 M, its concentration
after exercise, but also even at higher concentrations. We also
examined the effects of cAMP (evoked by forskolin), PKC (activated by
PMA), and intracellular Ca2+
(evoked by A-23187) on the -AR mRNA level in vitro, but none of
these intracellular factors had increased mRNA levels after 30 min of
incubation. Thus it appears that other mechanisms underlie the
exercise-induced elevation of -AR mRNA level in human lymphocytes.
Glucocorticoids pass through the plasma membrane and nuclear membrane
and bind to receptors located in the nuclear space. In other words,
glucocorticoids possess a signal-transduction system that is
independent of second-messenger recruitment. Human 2-ARs contain the
glucocorticoid response element sequence in the 5-flanking
region of its gene (10, 20). Glucocorticoids have been found to
increase the 2-AR number and
steady state of 2-AR mRNA
levels in several cell lines (8, 11, 16), and increased accumulation of
2-AR mRNA was detected 15 min
after exposure to dexamethasone (24). We therefore investigated the relationship between changes in plasma cortisol concentration and
changes in -AR mRNA level. However, there was little increase in plasma cortisol concentration in response to exercise, and the data
obtained failed to show any relationship between changes in plasma
cortisol concentration and changes in -AR mRNA level. This suggests
that glucocorticoids are not a major factor in the upregulation
of -AR mRNA levels during dynamic exercise.
Maisel et al. (23) pointed out that a change in lymphocytic subset
composition contributes to the increase in number of lymphocytic
-ARs. They demonstrated that dynamic exercise markedly increases the
number of T suppressor/cytotoxic and
natural killer cells but only modestly increases the number of T helper
cells; T helper cells have few -ARs, whereas T
suppressor/cytotoxic and natural killer
cells have a high -AR content. They also found no significant
alteration in the number of -ARs in subsets other than natural
killer cells after exercise, suggesting that the exercise-induced
increase in -AR of mixed lymphocytes is caused largely by
redistribution of circulating cell subsets that differ in -AR
number. They also reported that the observed redistribution of
circulating subsets alone can only explain an apparent increase in
-AR of 19% in mixed lymphocytes (23). In general, maximal exercise
induces a near doubling of lymphocytic -ARs, as found in the present
study. Thus it seems impossible to explain the observed increase in
-AR number in mixed lymphocytes without postulating an increase in
the number of -ARs on individual cells (13, 25). In fact, Ratge et
al. (29) found that the numbers of -ARs on T cells, B cells, and
monocytes are increased by dynamic exercise.
Van Tits et al. (33) demonstrated an important role of the spleen as a
marginal pool of lymphocytes that releases lymphocytes in response to
isoproterenol infusion and suggested that the lymphocytes pooled in the
spleen are relatively rich in -ARs, compared with lymphocytes in the
peripheral blood. They also suggested the possibility that the acute
increase in -AR number induced by dynamic exercise may be mainly due
to release of lymphocytes with a higher -AR content from the spleen
into the circulation. In their report, the increase in lymphocyte
-AR number induced by isoproterenol infusion was attenuated by 40%
in splenectomized patients compared with control subjects. However, in
their experiment on splenectomized patients, isoproterenol infusion
still increased the -AR number by ~500 sites/cell in mixed
lymphocytes compared with before infusion, with no change in
lymphocytic subset composition except for an increase in natural killer
cells (from 3.6 to 8.4% of mixed lymphocytes). If this increase were
explained by an increase in natural killer cells released from
secondary lymphoid organs alone, the natural killer cells would have to
have >10,000 sites/cell of -AR. In general, natural killer cells
have only 2,000-3,000 sites/cell of -AR, even after exercise
(23). These facts, together with the data reported by Maisel et al.
(23), suggest that part of the increase in -AR number and mRNA level
in mixed lymphocytes induced by dynamic exercise is caused by
upregulation of -ARs on single cells. However, we were unable to
identify the mechanisms underlying the exercise-induced upregulation of
-AR in this study, and other mechanisms that may elevate the -AR
mRNA level during exercise, such as the tyrosine kinase pathway related
to growth factors and cytokines, signals from extracellular matrix, and shear stress due to the increase in blood velocity, should be assessed
in the future.
It is unclear whether exercise-induced changes in -AR number and
mRNA level in lymphocytes run parallel with changes in other organs.
Acute treadmill running increased the -AR number in the sarcolemmal
membranes of rat myocardium (19), as well as in lymphocytes in the
present study. However, there was no change in the -AR number in rat
skeletal muscles after treadmill running (4). It is known that
expression of some receptors is regulated in a tissue-specific manner.
For example, thyroid hormones increase the
2-AR mRNA level in heart and
lung but decrease the level in the liver (21). Therefore,
exercise-induced increase in -AR number and mRNA level may be a
tissue-specific event. Friedman et al. (14) reported that 1-h treadmill
running decreased chronotropic responsiveness to isoproterenol infusion
in intact dogs. Martin et al. (26) also observed that there was no
effect induced by a single 1-h period of treadmill running on human
cardiovascular -AR responses measured during graded-dose
isoproterenol infusion. These observations are inconsistent with the
report by Izawa et al. (19), in which treadmill running increased
-AR number and adenylyl cyclase activity in sarcolemmal membranes of
rat myocardium. The cause of this discrepancy is unclear. However, the
exercise-induced increase in -AR number and adenylyl cyclase
activity in lymphocytes began to decrease after exercise and fell to a
level below the baseline within 30 min (5). Therefore, if such a change
also occurs in the heart, the prolonged duration for determining the responsiveness of the cardiovascular system to -AR-agonist infusion after exercise might cause this inconsistency. It also appears likely
that the differing results of these studies reflect a species variation
in the effects of acute exercise on the cardiac -AR system.
In conclusion, by using competitive RT-PCR analysis, we demonstrated
that dynamic exercise-induced increases in the -AR number on human
lymphocytes is accompanied by an increase in -AR mRNA level. The
rapid increases in -AR mRNA level may be an early adaptive response
to increasing sympathomimetic effects during dynamic exercise. The
nature of the trigger responsible for upregulation of -AR during
exercise, however, remains unknown.
ACKNOWLEDGEMENTS
We thank Dr. Junji Ohnishi for help in constructing the -AR
competitor for competitive reverse transcription-polymerase chain reaction analysis and Dr. Yoshiharu Nabekura for managing the subjects.
FOOTNOTES
Address for reprint requests: H. Miyazaki, Gene Experiment Center, Univ. of Tsukuba, Tsukuba-City, Ibaraki 305, Japan.
Received 4 March 1996; accepted in final form 12 February 1997.
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