(Hypertension. 1999;34:e5.)
© 1999 American Heart Association, Inc.
Letters to the Editor - Web |
Adrenergic Receptor Subtypes in Human Peripheral Blood Lymphocytes
Department of Pediatric Immunology, Wilhelmina Children’s Hospital of the University Medical Center Utrecht, Utrecht, The Netherlands
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To the Editor:
Ricci et al1 reported that human
peripheral blood lymphocytes express
1-adrenergic receptor subtypes. The authors
present results of radioligand binding assays with
[3H]-prazosin as the radioligand for
1-adrenergic receptors, showing that the
maximal density of the binding sites is 175 fmol per
106 cells. This would mean that the actual number
of receptors per cell would be exactly 100 000, which is an unusually
high number of receptors. Cell lines that are known for their high
expression of 1-adrenergic receptors have been
reported to express 10 000 to 25 000 receptors per
cell.2 3 We have to keep in mind, however, that these
numbers are found on kidney or smooth muscle cells, which are much
larger than the freshly isolated lymphocytes that were used in the
study by Ricci. Moreover, it is known for other G-protein–coupled
receptors, eg, ß2-adrenergic receptors, that
are expressed on lymphocytes that the actual number of receptors per
cell is around 2000.4 Thus, the number reported by Ricci
et al for 1-adrenergic receptors on
lymphocytes is extraordinary high.
The authors do cite the literature on
1-adrenergic receptor expression in
peripheral blood lymphocytes only partly correct. The only
study that reports 1-adrenergic receptor
binding on lymphoid cells from peripheral blood is the
study by Jetschmann et al.5 However, these authors only
examined NK cells where they reported the presence of 600 to 700
receptors per cell. Casale at al6 could not demonstrate
binding sites on peripheral blood lymphocytes, whereas
Maestroni et al7 only investigated
1-receptor expression in bone marrow cells and
not in peripheral blood lymphocytes.
More importantly, the authors claim that they can displace ligand from
the receptor by the use of antibodies specific to the three receptor
subtypes. Theoretically, this is a good approach that could give
information on the presence of specific receptor subtypes. However,
this approach requires antibodies that bind to the ligand-binding site
of the receptor so that competition for the same site can be expected.
Unfortunately, such antibodies are not available. The antibodies used
in the study by Ricci et al recognize intracellular domains of the
receptor and do not bind to the ligand binding domains of the
1-receptor subtypes. We have to conclude that
these antibodies cannot be used in binding competition experiments,
because they will not compete for binding to the same site as the
ligand. In addition, it is highly unlikely that these antibodies would
bind to the intracellular domains to the receptors in intact cells,
since large molecules such as antibodies do not enter the cell.
Another point of concern deals with the way cells are handled and the amount of cells used in the assays. In the Methods section, it is stated that 40 mL of blood was drawn from each donor. Subsequently, 10x106 cells were used for RNA isolation. The other cells were frozen at -80°. For their binding studies, it was reported that 1x106 cells per point were used and that all studies were performed in triplicate. The authors tested 9 concentrations of tritiated ligand both in the presence and absence of unlabeled ligand in order to determine aspecific binding. This part of the study would require 54x106 cells. In addition, inhibition by three different antibodies was tested, using 8 concentrations of each antibody, which would require an additional 72x106 cells. In general, with the use of 40 mL of blood, it is possible to isolate about 40x106 cells. Thus, it is not clear how all the experiments could be performed with the indicated amount of blood.
When we then look at the PCR data, there is a discrepancy between the
results of the RT-PCR analysis and the
autoradiographic data, especially with respect to 1B.
The results in panel A for 1B suggest that in lane 2, the signal is
by far the strongest; whereas in panel B, this is the weakest
signal.
In summary, we think that in view of the technical setup of the
experiments, the conclusion that freshly isolated human
peripheral blood lymphocytes express
1-adrenergic receptors is not warranted by
this study.
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References |
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TopIntroductionReferencesIntroduction References |
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1. Ricci A, Bronzetti E, Conterno A, Greco S, Mulatero P, Schena M, Schiavone D, Tayebati SK, Veglio F, Amenta F.
1-Adrenergic receptor subtypes in
human peripheral blood lymphocytes.
Hypertension. 1999;33:708–712.
2.
Yang M, Büscher R, Taguchi K, Grübel B,
Insel PA, Michel MG. Protein kinase C does not mediate
phenylephrine-induced down-regulation of Madin Darby
canine kidney cell alpha-1B adrenoceptors. J Pharmacol Exp
Ther. 1998;286:36–43.
3. Colucci WS, Akers M, Wise GM. Differential effects of norepinephrine and phorbol ester on alpha-1 adrenergic receptor number and surface-accessibility in DDT1MF-2 cells. Biochem Biophys Res Commun. 1988;156:924–930.[Medline] [Order article via Infotrieve]
4. Fratelli M, Gagliardini V, De Blasi A. Low affinity of adrenergic receptors for agonists on intact cells is not due to receptor sequestration. Biochem Biophys Acta. 1989;1012:178–183.[Medline] [Order article via Infotrieve]
5.
Jetschmann J-U, Benschop RJ, Jacobs R, Kemper A,
Oberbeck R, Schmidt RE, Schedlowski M. Expression and in-vivo
modulation of - and ß-adrenoceptors on human natural killer
(CD16+) cells. J Neuroimmunol. 1997;74:159–164.[Medline]
[Order article via Infotrieve]
6. Casale TB, Kaliner M. Demonstration that circulating human blood cells have no detectable alpha1-adrenergic receptors by ligand binding analysis. J Allergy Clin Immunol. 1984;742:812–819.
7. Maestroni GJ, Conti A. Noradrenergic modulation of lymphohematopoiesis. Int J Immunopharmacol. 1994;16:117–122.[Medline] [Order article via Infotrieve]
Response
Department of Cardiovascular and Respiratory Sciences, University La Sapienza, Rome, Italy
Department of Medicine and Experimental Medicine, University of Turin, Turin, Italy
Section of Human Anatomy, Department of Pharmacological Sciences and Experimental Medicine, University of Camerino, Camerino, Italy
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A recent paper of our group,1 reported that human peripheral blood lymphocytes (HPBL) express
1-adrenergic receptor subtypes. Our
conclusions are strongly criticized by Drs Kavelaars, Ruppe von der
Voort, and Heijnen. Their opinion is that in view of the technical
setup of the experiments, it is not demonstrated that freshly isolated
HPBL express 1-adrenergic receptors.
We do not agree with these criticisms for the reasons detailed below.
First, the problem of a rather high density of
[3H]prazosin binding sites in HPBL was already
discussed in our manuscript. These criticisms were derived mainly from
wrong calculations. It is not true that cell lines express at most 4 to
10 times fewer receptors than HPBL in our study. Transfected cell lines
were reported to bind 1-adrenergic
radioligands in the range between 2856 to 4740 fmol/mg
protein.2 3 The mean protein concentration of preparations
of HPBL is 1 mg/2.7 to 3x107
cells.4 Hence, our preparations of HPBL have an
1-adrenergic receptor concentration similar to
that reported for transfected cell lines. Second, references to
literature used in our paper were correct with the exception of the
quotation of Reference 7 instead of 10 and of Reference 10 instead of 9
in the 2nd paragraph of the Discussion. We were also surprised that in
spite of concerns for the presence of
1-adrenergic receptors in HPBL, Kavelaars et
al apparently ignored two recent papers,5 6 which were not
published when our work was accepted. In these studies,
1-adrenergic receptors were demonstrated by
analyzing lymphocyte catecholamine response5
and the amiloride-sensitive sodium channels.6 Third, in
our analysis of [3H]prazosin, binding
was performed by using two different approaches: (1) conventional
radioligand binding assay with compounds selective for
1-adrenergic receptor subtypes, and (2)
antireceptor subtype antibodies. Apparently, the
radioligand binding part of the study was not considered by
Kavelaars et al. Their criticisms were on experiments with antibodies.
Of course, we are unable to establish if and to which extent the
antibodies used recognize 1-adrenergic
receptor subtypes binding domain. However, the explanation of Kavelaars
et al is simplistic as we cannot exclude the occurrence of interactions
(indirect) between the antibody and the receptor binding domain after
blockade of a portion of receptor sequence with a specific antibody. In
line with this hypothesis are binding curves generated in the presence
of antibodies. On the other hand, experiments with antibodies are just
one of the different approaches for demonstrating
1-adrenergic receptor subtypes. Our assumption
of the presence of 1-adrenergic receptor
subtypes in HPBL was also confirmed by some recent work using
cytospin-centrifuged HPBL and immunocytochemistry. In these
experiments, we observed that 25% of HPBL were immunoreactive for
1A receptor protein, 40% for
1B receptor protein, and 22% for
1D receptor protein. Fourth, on the amounts of
blood and lymphocytes, Kavelaars et al failed to consider that we
collected blood from 30 subjects. We did not use blood from 30 subjects
for all experiments; we used a single subject for a series of complete
experiments to obtain reliable data for a given parameter.
Standard deviations reflect intersubject variability. Fifth, concerning
PCR data, note that our protocol did not include quantitative PCR.
Lanes show just the presence of signals, whereas quantitative
analysis was based on radioligand binding assay
data as indicated in our paper. 1 Moreover, in Figure 1
(1B receptor panel),
autoradiography improved detection of a signal affected
by interfering factors only in one subject. Really, we do not
understand the purpose of these criticisms, which appear
inappropriate.
In summary, with the exception of some imprecision in quoting
references, criticisms of our study by Kavelaars et al do not seem
based on justified methodological problems. Our work, as well as more
recent evidence,5 6 indicate that HPBL express
1-adrenergic receptors. A crucial point to
investigate in future studies is the significance of
1-adrenergic receptors.
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References |
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TopIntroductionReferencesIntroduction References |
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1. Ricci A, Bronzetti E, Conterno A, Greco S, Mulatero P, Schena M, Schiavone D, Tayebati SK, Veglio F, Amenta F.
1-Adrenergic receptor subtypes in
human peripheral blood lymphocytes.
Hypertension. 1999;33:708–712.
2.
Faure C, Pimoule C, Arbilla S, Langer SZ, Graham D.
Expression of 1-adrenoceptor subtypes in
rat tissues: implications for 1-adrenoceptor
classification. Eur J Pharmacol .1994;268:141–149.
3.
Goetz AS, Lutz MW, Rimele TJ, Saussy DL.
Characterization of 1-adrenoceptor
subtypes in human and canine prostate membranes. J Pharmacol
Exp Ther.. 1994;271:1228–1233.
4. Meurs H, van der Bogaard W, Kauffman HF, Bruynzeel PLB. Characterization of (-)-[3H]dihydroalprenolol binding to intact and broken cell preparations of human peripheral blood lymphocytes. Eur J Pharmacol.. 1982;85:185–194.[Medline] [Order article via Infotrieve]
5. Baerwald CG, Whale M, Ulrichs T, Jonas D, von Bierbrauer A, von Wichert P, Burmester GR, Krause A. Reduced catecholamine response to lymphocytes from patients with rheumatoid arthritis. Immunobiology.. 1999;200:77–91.[Medline] [Order article via Infotrieve]
6.
Bubien JK, Cornwell T, Bradford AL, Fuller CM, Duvall MD,
Benos DJ. -Adrenergic receptors regulate human lymphocyte
amiloride-sensitive sodium channels. Am J Physiol.. 1998;275:C702–C710.
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