(Hypertension. 1999;33:708-712.)
© 1999 American Heart Association, Inc.
Scientific Contributions |
1-Adrenergic Receptor Subtypes in Human Peripheral Blood Lymphocytes
From the Department of Cardiovascular and Respiratory Sciences (A.R., E.B, S.G.), "La Sapienza" University, Rome; Department of Medicine and Experimental Oncology, Chair of Internal Medicine (A.C., P.M., M.S., D.S., F.V.), University of Turin; Section of Human Anatomy, Department of Pharmacological Sciences and Experimental Medicine (S.K.T., F.A), University of Camerino, Italy.
Correspondence to Francesco Amenta, MD, Dipartimento di Scienze Farmacologiche e Medicina Sperimentale, Via Scalzino, 3-62032 Camerino, Italy. E-mail amenta{at}cambio.unicam.it
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Abstract |
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Abstract—We investigated the expression of
1-adrenergic receptor subtypes in intact
human peripheral blood lymphocytes using reverse
transcription–polymerase chain reaction (RT-PCR) and
radioligand binding assay techniques combined with
antibodies against the three subtypes of 1-adrenergic
receptors (1A, 1B, and
1D). RT-PCR amplified in peripheral blood
lymphocytes a 348-bp 1A-adrenergic receptor fragment, a
689-bp 1B-adrenergic receptor fragment, and a 540-bp
1D-adrenergic receptor fragment.
Radioligand binding assay with [3H]prazosin
as radioligand revealed a high-affinity binding with a
dissociation constant value of 0.65±0.05 nmol/L and a maximum density
of binding sites of 175.3±20.5 fmol/106 cells. The
pharmacological profile of [3H]prazosin binding to human
peripheral blood lymphocytes was consistent with
the labeling of 1-adrenergic receptors. Antibodies
against 1A-, 1B-, and
1D-receptor subtypes decreased
[3H]prazosin binding to a different extent. This
indicates that human peripheral blood lymphocytes express
the three 1-adrenergic receptor subtypes. Of the three
different 1-adrenergic receptor subtypes, the
1B is the most represented and the
1D, the least. Future studies should clarify the
functional relevance of 1-adrenergic receptors expressed
by peripheral blood lymphocytes. The identification of
these sites may represent a step for evaluating whether they
represent a marker of 1-adrenergic receptors in
cardiovascular disorders or for assessing responses to
drug treatment on these receptors.
Key Words: lymphocytes • receptors, adrenergic, alpha • receptor subtypes • receptor antibodies
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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In the past few years,
- and ß-adrenergic receptors
have been demonstrated in peripheral blood lymphocytes with
the use of radioligand binding assay techniques. The
majority of information is available on ß-adrenergic receptors, which
were investigated primarily in essential hypertension, impaired left
ventricular function, and acute stress.1 2 3 4 5
Data on the expression of -adrenergic receptors in
peripheral blood lymphocytes are less extensive, with the
2-receptor subtype being the most extensively
investigated.6 7 Some studies on the
1-adrenergic receptor have not found its
expression in human peripheral blood
lymphocytes8 9 and some studies
have.10
1-Adrenergic receptors mediate some
cardiovascular sympathetic responses, such as
arteriolar smooth muscle constriction11 and cardiac
contractility,12 and are involved in a
variety of cardiovascular disorders, including
hypertension, heart failure, and cardiac
hypertrophy.13
1-Adrenergic receptors represent a
class of heterogeneous receptors.14 Cloning
studies have identified three distinct
1-adrenergic receptor subtypes, named
1a-, 1b-, and
1d-adrenergic receptors.14 15 16 17 At
present, 1-adrenergic receptor subtypes
are defined as 1A
(1a), 1B
(1b), and 1D
(1d), with uppercase and lowercase subscripts
being used to designate native or recombinant receptor,
respectively.14 18
In view of the difficulty of investigating
1-adrenergic receptors in vivo or in vitro
using human samples of cardiovascular system,
circulating blood cells may represent a model for the study of
the cardiovascular
1-adrenergic receptor system.
In this article we have characterized
1-adrenergic receptor subtypes expressed by
human peripheral blood lymphocytes by reverse
transcription–polymerase chain reaction (RT-PCR) to detect mRNA
expression and by radioligand binding assay techniques
combined with antibodies against different subtypes of
1-adrenergic receptors.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Subjects and Peripheral Blood Lymphocyte Separation
Blood samples (40 mL) were drawn from 30 healthy subjects (15 men and 15 women; age, 26 to 48 years), after informed consent had been obtained, for peripheral blood lymphocyte isolation. Cells were diluted 1:3 with phosphate-buffered saline (0.85% NaCl and 6.7 mmol/L potassium phosphate, pH 7.2) and separated on a Ficoll-Hypaque gradient. The layer of mononuclear cells was removed and resuspended. Cells (10x106) were sedimented by centrifugation in Eppendorf tubes, lysed with 1 mL RNAfast by repetitive pipetting, and then stored at -80°C until further analysis. Cells used for radioligand binding assay after separation were collected in polypropylene tubes and immediately frozen at -80°C.
Reverse Transcription–Polymerase Chain Reaction
Total RNA was extracted using the RNAfast method. RT was
performed using 1 µg total RNA, according to the manufacturer's
instructions (Perkin-Elmer). PCR was carried out in an automated DNA
thermal cycler (Perkin-Elmer) using the following specific
primers: 1A sense and antisense,
5'-ACTACATCGTCAACCTGGCG-3' and 5'-TGATCTGGCAGATGGTCTCG-3',
respectively; 1B sense and antisense,
5'-TCGGTGGCCTGCAACCGGCACCTG-3' and 5'-ATGCCCAAGGTTTTGGCTGCTTTCTT-3',
respectively; 1D sense and antisense,
5'-GTGGTGAGTGCTCAGGGCGTG-3' and 5'-GATGACCGCCATGGGCAGGT-3'. The
amplification protocol was 90 seconds at 94°C, 2 minutes at 60°C,
and 2 minutes at 72°C, with extension of 1 second per cycle for 30
cycles. PCR products were electrophoresed on a 1.5% agarose gel
containing 0.5 mg/mL ethidium bromide. After gel electrophoresis, the
amplified fragments of 1-adrenergic receptor
subtypes were blotted to nylon filters and hybridized with a specific
nested oligonucleotide for each subtype of
1-adrenergic receptor, and were labeled by T4
kinase with [
-32P]-ATP. Specific nested
oligonucleotides used for 1A,
1B, and 1D were
5'-AACCATCGTCACCCAGAGGA-3', 5'-ATGGAACTCCTGGGGTTGTG-3', and
5'-CTTATGGCCGTGGCAGGTAA-3', respectively. A specific activity of
4x106 cpm/mL was added to the hybridizing
solution (5x SSPE, 0.5x SDS, 5x Denhardt's solution), and the
filters were incubated for 2 hours at 42°C. Filters were then washed
twice in 2x SSPE at room temperature and once in 5x SSPE at 52°C
and exposed for 24 to 48 hours at –80°C.
Radioligand Binding Assay
Three hundred microliters of a lymphocyte suspension containing
1x106 cells was incubated in triplicate for 60
minutes at 25°C with increasing concentrations of
[3H]prazosin (0.05 to 4 nmol/L) in 50
mmol/L Tris-HCl containing 150 nmol/L NaCl, 5 mmol/L EDTA,
100 µmol/L pargyline, and 3 µmol/L ascorbic acid (pH
7.4). Nonspecific binding was defined by incubating lymphocytes with
[3H]prazosin in the presence of 10
µmol/L phentolamine.
The pharmacological specificity of [3H]prazosin binding to human peripheral blood lymphocytes was assessed by incubating suspensions containing 1x106 cells with 0.5 nmol/L [3H]prazosin in the presence of increasing concentrations (0.001 nmol/L to 1 mmol/L) of compounds active on adrenergic receptors, dopamine, and serotonin receptors.
At the end of incubation, cells were isolated onto Whatman GF-B glass fiber filters with the use of a manifold filtration apparatus. Filters were washed rapidly twice with ice-cold incubation buffer and transferred into scintillation vials for radioactivity measurement.
Experiments With Antibodies Against 1-Adrenergic
Receptor Subtypes
Suspensions of peripheral blood lymphocytes
(1x106 cells/mL) were preincubated for 1 to 2
hours at 25°C with increasing concentrations (0.05 to 2 µg/mL) of
goat polyclonal immunoglobulins raised against
1A-, 1B-, and
1D-adrenergic receptors. Immunoglobulins were
used alone, in association (anti-1A and
anti-1B, anti-1A and
anti-1D, anti-1B and
anti-1D), and all together. For control
purposes, peripheral blood lymphocytes were incubated with
immunoglobulins preadsorbed with 10 µg/mL of synthetic peptides used
for raising antibodies. After preincubation,
[3H]prazosin binding was assayed according to
the above protocol with a radioligand concentration of 0.5
nmol/L.
Data Analysis
Data from binding and competition experiments were calculated
with the Radlig program.19 In experiments performed with
antibodies against 1-adrenergic receptor
subtype, [3H]prazosin binding in the presence
of preabsorbed antibody was considered as total binding. Binding values
obtained in the presence of antibodies against
1A-, 1B-, and
1D-adrenergic receptor concentrations causing
maximal inhibition of radioligand binding to human
peripheral blood lymphocyte (0.5 µg/mL) were considered
to represent nonspecific retention of radioligand
to lymphocyte preparation or to be the result of labeling sites other
than 1-adrenergic receptors. The
difference between total binding and nonspecific
radioligand retention was considered as the specific
binding defined immunochemically.
Chemicals
[3H]prazosin (specific activity, 78.0
Ci/mmol) was purchased from the Amersham Radiochemical Center.
Polyclonal anti-1A– (No. Sc-1474),
anti-1B– (No. Sc-1476), and
anti-1D– (No. Sc-1477) adrenergic
receptor subtype immunoglobulins and 1A- (No.
Sc-1474P), 1B- (No. Sc-1476P), and
1D- (No. Sc-1477P) blocking peptides were
purchased from Santa Cruz Biotechnology. Immunoglobulins used did not
cross-react with other - or ß-adrenergic receptor subtypes (data
not shown). (-)-trans-Mephendioxan20 was
a gift of Dr. W. Quaglia of the University of Camerino. Other chemicals
were obtained from Sigma Chemical Co. and Research
Biochemicals.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Reverse Transcription–Polymerase Chain Reaction
RT-PCR amplified a 348-bp
1A-adrenergic
receptor fragment, a 689-bp 1B-adrenergic
receptor fragment, and a 540-bp 1D-adrenergic
receptor fragment (Figure 1, panels labeled A). 1B-Adrenergic receptor mRNA
was expressed with higher abundance than 1A-
and 1D-adrenergic receptor mRNAs (Figure 1, panels labeled A). The agarose gel was then blotted onto a
nylon filter and hybridized with the nested
oligonucleotides to increase the sensitivity of
detection (Figure 1, panels labeled B).
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Radioligand Binding Assay
[3H]Prazosin was bound specifically to
human peripheral blood lymphocytes. The binding was
concentration dependent, with a Kd value of
0.65±0.05 nmol/L (Figure 2A) and
Bmax of 175.3±20.5
fmol/106 cells (Figure 2B).
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), 
), and
) receptor immunoglobulins on
[3H]prazosin (at concentration of 0.5 nmol/L) binding to
human peripheral blood lymphocytes (C). A, Cells were
incubated with increasing concentrations of the radioligand
alone (total binding, 
). Specific binding
values (
) did not
affect [3H]prazosin binding to human
peripheral blood lymphocytes. Data of
[3H]prazosin specifically bound are expressed femtomoles
per 106 cells. Points are mean±SEM of triplicate
determinations.
Analysis of data on the pharmacological profile of
[3H]prazosin binding to human
peripheral blood lymphocytes showed that the most powerful
displacers of [3H]prazosin were
antagonists of different subtypes of
1-adrenergic receptors
(spiperone>(-)-trans-mephendioxan>(+)-niguldipine>5-methyl-urapidil)
or nonselective 1- or -adrenergic receptor
antagonists (prazosin>phentolamine)
(Table). The 2-adrenergic
receptor agonist clonidine, norepinephrine, the
ß-adrenergic receptor antagonist pindolol, and compounds
active on dopamine (dopamine) and serotonin receptors
(serotonin and methysergide) (Table) were much less
potent than compounds with an -adrenergic receptor profile or were
ineffective as displacers of [3H]prazosin
binding.
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Anti–1A-, anti–1B-,
and anti–1D-adrenergic receptor
immunoglobulins decreased [3H]prazosin binding
to human peripheral blood lymphocytes progressively from an
immunoglobulin concentration of 0.1 to 0.2 µg/mL (Figure 2C).
The maximal inhibition of [3H]prazosin binding
was observed at an immunoglobulin concentration of 0.5 µg/mL (Figure 2C). No further decrease of binding was noticeable at higher
immunoglobulin concentrations (Figure 2C). Analysis of
inhibition of [3H]prazosin binding by single
antibodies against 1-adrenergic receptor
subtypes or by immunoglobulins in association showed that about 28% of
sites labeled by [3H]prazosin corresponded to
1A-adrenergic receptors, 40% to
1B-adrenergic receptors, and 18% to
1D-adrenergic receptors (Figure 2C).
Immunoglobulins preadsorbed with synthetic peptides used for raising
antibodies did not affect [3H]prazosin binding
to human peripheral blood lymphocytes (Figure 2C).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Elevated sympathoadrenal tone and imbalance of
- and
ß-adrenergic receptor function are important factors in the
pathogenesis of essential hypertension.21
1-Adrenergic receptors are important mediators
of the sympathetic neuroeffector system,12 mediate
positive inotropic effects of epinephrine in rat
ventricular strips, and participate in vascular smooth
muscle tone regulation.21 22 23 -Adrenergic receptor
activation contracts isolated blood vessels and increases blood
pressure in vivo.21 22 23 On the basis of these observations
-adrenergic receptor blockade was introduced for the treatment of
hypertension.18 The development of selective
1-adrenergic receptor antagonists
(prazosin and its derivatives) has led to the widespread use of these
drugs as antihypertensive agents for many years.18
The 1-adrenergic receptor subtype responsible
for vascular contraction may depend on both the vascular bed and the
species investigated.12 18 The
1-adrenergic receptor subtype(s) responsible
for blood pressure regulation in the intact individual has been not
identified yet. The most probable reasons for the limited amount of
information on the topic are the complexity of investigating
sympathetic cardiovascular activity in vivo and the
difficulty of obtaining samples of human vascular tissue suitable for
pharmacological and functional analysis in vitro.
It has been hypothesized that analysis of
peripheral blood lymphocyte adrenergic receptor expression
may represent a model for studying the
cardiovascular adrenergic receptor
system.1 2 3 4 5 6 7 The suitability of techniques so far proposed
for demonstrating -adrenergic receptor expression in normal lymphoid
cells is still controversial. 2-Adrenergic
receptors were demonstrated in human peripheral blood
lymphocytes using [3H]yohimbine and
[3H]clonidine as
radioligands.6 7 Radioligand
binding studies performed with [3H]prazosin
were unable to identify 1-adrenergic receptors
in normal8 or leukemic human lymphocytes7 or
reported the expression of these receptors in peripheral
human natural killer cells.10 In view of these
discrepancies we have reinvestigated the topic using both molecular
biology and radioligand binding assay techniques.
Currently, truly selective agonists or antagonists for
discriminating 1-adrenergic receptor subtypes
are not available.18 In view of this, we used antibodies
against 1A-, 1B-, and
1D-adrenergic receptor subtypes for
investigating the receptor subtypes labeled by
[3H]prazosin in blood lymphocytes.
The present study has shown, with the use of different
techniques, such as RT followed by nested PCR and
radioligand binding assay in association with specific
antibodies against 1-adrenergic receptor
subtypes, that human peripheral blood lymphocytes express
the three 1-adrenergic receptor subtypes so
far identified (1A,
1B, and 1D). The
density of 1-adrenergic receptors assayed in
the present study is rather high (175.3±20.5
fmol/106 cells). This suggests that human
peripheral blood lymphocyte
1-adrenergic receptors may have functional
relevance. Comparative analysis of the density of
1-adrenergic receptors measured in this work
with data reported for lymphocyte 2-adrenergic
receptors6 7 showed a ratio of 1-
to 2-adrenergic receptor of approximately 7.9
in human peripheral blood lymphocytes. The role of
lymphocyte 1-adrenergic receptors has not been
clarified yet. It has been suggested that
1-adrenergic receptors modulate
lymphohematopoiesis.10 The
1-adrenergic receptor antagonist
prazosin enhances myelopoiesis and decreases the number of thymocytes
and of splenic T and B lymphocytes.10 Future studies
should clarify the possible roles of the different receptor subtypes on
lymphocyte function.
The association of [3H]prazosin
radioligand binding assay with antibodies raised against
the different 1-adrenergic receptor subtypes
has shown that 1B receptor is the
1-adrenergic receptor subtype most
represented in human peripheral blood
lymphocytes and 1D receptor, the least. These
radioligand binding data are consistent with
semiquantitative analysis of mRNA levels tested by RT-PCR.
Future studies should clarify the relevance of this uneven density of
1-adrenergic receptor.
In view of the involvement of 1-adrenergic
receptors in the regulation of cardiovascular
homeostasis,12 1-adrenergic
receptor subtype expression in human peripheral blood
lymphocytes may represent a marker of the status of
1-adrenergic receptors in
cardiovascular disorders or may be useful for
monitoring responses of these receptors to drug treatment. In
line with this hypothesis are our preliminary results suggesting a
downregulation of lymphocyte 1-adrenergic
receptors in patients with essential hypertension.
Received July 31, 1998; first decision September 1, 1998; accepted October 6, 1998.
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References |
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