![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 298, Issue 2, 441-452, August 2001
-Adrenoceptor Subtypes by Smooth Muscle Cells
and Adventitial Fibroblasts in Rat Aorta and in Cell Culture
Department of Cell and Molecular Physiology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina
 |
Abstract |
|---|
 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Previous radioligand binding reports of vascular 
-adrenoceptor (AR)
density have been limited to total 1- or
2-ARs. Studies using whole blood vessel homogenates have
not differentiated among receptor or mRNA expression by medial smooth
muscle cells (SMCs) versus adventitial fibroblasts (AFBs). Therefore,
we used quantitative reverse transcription-polymerase chain reaction
and radioligand binding to measure -AR subtypes in media,
adventitia, and cultured SMCs and AFBs from rat aorta. Both media and
adventitia expressed 1A-, 1B-,
1D-, and 2D-AR mRNAs, but in markedly
different abundances. Total 1-AR density was the same
for media and adventitia (Bmax = 101 ± 10 versus 96 ± 16 fmol/mg of protein). However, densities for 1A-, 1B-, and
1D-AR subtypes in media were 19 ± 2, 26 ± 4, and 55 ± 2%, and in adventitia were 44 ± 3, 37 ± 5, and 19 ± 2%. No 2B- or 2C-AR
transcripts were detected in either layer or in cultured SMCs or AFBs.
Total 1-AR densities in cultured SMCs and AFBs
(Bmax = 111 ± 4 and 48 ± 6 fmol/mg of protein, respectively) were similar to media and adventitia,
with 1B- and 1D-AR transcript levels and
receptors largely sustained. However, 1A- and
2D-AR expression in cultured SMCs and AFBs was strongly
reduced, compared with media and adventitia, an effect not prevented by
30 different culture conditions. Like SMCs, exposure of AFBs to
norepinephrine induced protein synthesis and proliferation of AFBs.
This is the first study to quantitate -AR subtype expression in
media and adventitia and in cultured SMCs and AFBs. In addition, we
report the intriguing finding that AFBs express 1-ARs in
similar abundance as medial SMCs and that norepinephrine induced them to proliferate.
| |
Introduction |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Vascular
-adrenoceptors (ARs) mediate sympathetic regulation of smooth muscle
cell (SMC) contraction for control of blood flow, pressure, and
vascular wall compliance. These receptors may also regulate growth of
vascular wall cells, although much less is known about this
possibility. Determination of which -AR subtypes are expressed and
mediate constriction of different vessels was recently reviewed
(Piascik et al., 1996
; Docherty, 1998). Among the three
1- (1A, 1B, and 1D) and three
2-ARs (2D, 2B, and 2C) cloned and expressed by
rat tissues, a single -AR subtype often subserves sympathetic
regulation, e.g., the 2D/A-AR on pancreatic
islet cells or the 1B-AR on hepatocytes. In
contrast, blood vessels possess multiple subtypes, where the relative
subtype mRNA levels and receptor(s) mediating SMC contraction can vary among different vessels and species. Controversy has arisen from reports where mRNAs for three or more -AR subtypes have been detected in many arteries (e.g., aorta), yet not all of the subtypes appear "functional", as defined by constriction (Piascik et al., 1996; Docherty, 1998). However, recent studies demonstrate that norepinephrine induces growth of rat aorta SMCs in both cell and organ
culture by stimulation of an 1-AR subtype that
may not signal constriction (Chen et al., 1995; Xin et al., 1997). The importance of defining the distribution and function of vascular -AR
subtypes is further emphasized by evidence that changes in their
expression occur during maturation (Ibarra et al., 1997) and in
diseases such as hypertension (Villalobos-Molina and Ibarra, 1999) and
atherosclerosis (Handy et al., 1998).
Measurement of vascular -AR density by radioligand binding generally
has been confined to estimates of total 1- or
2-AR density because of lack of availability,
until recently (Docherty, 1998), of sufficiently selective subtype
antagonists, and because of difficulties in conducting binding studies
on small tissue samples. As well, subtype specific antibodies are not
available with sufficient selectivity to quantify receptor abundance in blood vessels (Hrometz et al., 1999; Shen et al., 2000). Reports of
mRNA levels have been limited mostly to qualitative determinations (Price et al., 1994; Rokosh et al., 1994; Piascik et al., 1996; Xu and
Han, 1996; Phillips et al., 1997; Docherty, 1998; Handy et al., 1998;
McNeill et al., 1999). In addition, previous reports of -AR
transcript and receptor abundance have been derived from whole vessel
homogenates and used to infer expression by SMCs. However, the arterial
wall is composed of three layers of different cell types: the intima is
composed of a single layer of endothelial cells (plus dispersed
"stellate" cells in mammals larger than rodents), the media is
composed of SMCs (and some non-SMC-like cells in mammals larger than
rodents) (Bochaton-Piallat et al., 1996: Faggin et al., 1999; Frid et
al., 1999), and the adventitia is composed of more than 99%
adventitial fibroblasts (AFBs) (cf. Chen et al., 1995; Faggin et al.,
1999) plus nerve endings and occasional mast cells and macrophages (and
a vasa vasorum that can extend into the outer media in animals larger
than the rabbit). Endothelial cells possess 2-
and possibly 1-ARs (Bockman et al., 1996;
Docherty, 1998), although the small number of these cells, compared
with other vascular wall cells, suggest they contribute minimally to
RNA or membrane preparations from the intact vessel wall. However, in
many arteries, the adventitia can be as thick as the media, with the
AFBs comprising a large fraction of the total vascular wall cells.
Perhaps because fibroblasts are regarded as structural cells and are
not known to express ARs, the possibility that AFBs might possess ARs
has not been questioned. Recently, however, we found that both media
and adventitia of the rat aorta express mRNAs for multiple
1- and 2-ARs (Yang et
al., 1999). However, quantitative mRNA transcript levels and receptor
proteins were not determined. In addition, little is known about the
effect of cell culture on -AR expression, despite its importance for
study of vascular -ARs.
Therefore, the purpose of this study was to develop quantitative RT-PCR
and radioligand binding assays to determine -AR subtype expression
in small tissue samples, i.e., rat aorta media and adventitia, and to
examine the effect of culture conditions on -AR expression by SMCs
and AFBs derived from these layers. A surprising finding was that AFBs
express the same four -ARs as media and with the same total
1-AR density, but where the proportion of AR
subtypes differs greatly between the two layers. Moreover, AFBs respond
to norepinephrine with proliferation and protein synthesis.
| |
Materials and Methods |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Tissue Collection.
As detailed elsewhere (Yang et al.,
1999), thoracic aortae of 300-g male Sprague-Dawley rats were isolated
under a pool of 4°C phosphate-buffered saline (PBS), after gently
opening the parietal pleura, separating away loose connective
tissue/fat, and transecting the segmental arteries at their origins.
Aortae were incubated for 25 min in a 37°C, 5%
CO2 incubator with 1 mg/ml collagenase, 1 mg/ml
soybean trypsin inhibitor, and 13.5 units/ml elastase (Worthington
Biochemicals, Freehold, NJ) to loosen endothelial cells and the
external elastic lamina. Adventitial and medial layers were then
separated with fine forceps in 4°C PBS, which contained 10 mM vanadyl
ribonucleoside complex (Life Technologies, Gaithersburg, MD) when
layers were being isolated for RNA extraction or the proteinase
inhibitors: 30 µl/ml aprotinin, 100 µg/ml phenylmethylsulfonyl fluoride, and 1 mM sodium orthovanadate (Sigma, St. Louis, MO) when
layers were being isolated for protein extraction. Endothelial cells
were removed by two strokes of a cotton-tipped applicator after opening
the media longitudinally. En-face staining with silver nitrate verified
removal of >99% of the endothelial cells. Layers were frozen in
liquid nitrogen and stored at 
80°C.
Smooth Muscle and Adventitial Fibroblast Cell Cultures.
Each
SMC and AFB primary culture was obtained from eight thoracic aorta
media and sterile-isolated adventitia as above from 200-g rats. SMCs
were dispersed from the media with the above enzymes, followed by
differential plating (the SMCs, but not the AFBs, readily adhere to
plastic culture dishes) to prevent contamination of SMC cultures with
any adherent AFBs (Eckhart et al., 1996). To obtain AFBs, adventitia
was minced into 1-mm2 pieces and incubated for 30 min at 37°C without shaking in 2.4 units/ml neutral protease II
(Roche Molecular Biochemicals, Summerville, NJ). After gentle
trituration, cells were placed in M199 culture media with 20% fetal
bovine serum (FBS) on ice to arrest protease activity. After repeating
this dispersion procedure three times, pooled cells were gently
resuspended in M199 plus FBS and sieved (38 µM, no. 400) to separate
the smaller AFBs from occasional non-AFBs present in adventitia
(SMC-like cells, mast cells, macrophages, and adipocytes). SMCs and
AFBs (20,000 cells/cm2) were grown in M199 + 10%
FBS, 200 mg/l L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin passaged at ~95% confluence with 0.10%
trypsin/EDTA every 3 (AFBs) or 5 days (SMCs), and seeded at a density
of 5000 cells/cm2. Unless noted otherwise, cells
were used in passages 3 to 5, carried 2 days beyond confluence, and
then growth-arrested for 2 days in serum-free defined medium,
consisting of 50% Dulbecco's modified Eagle's medium-high
glucose, 50% F12, 2.85 mg/l insulin, 5 mg/l transferrin, 35.2 mg/l ascorbic acid, 6 ng/l selenium, 100 units/ml penicillin, and 100 µg/ml streptomycin. As tests of culture homogeneity, absence of
endothelial cell contamination was confirmed with morphology and
anti-von Willebrand factor immunohistochemistry (Chen et al., 1995;
Eckhart et al., 1996). As well, macrophages, lymphocytes, and
endothelial cells cannot proliferate in the above culture conditions
sufficiently to survive first passage (Babij et al., 1993). Also, we do
not detect the SMC marker proteins, metavinculin, and SM1 in AFB
cultures that do, however, express fibroblast specific protein-1
(FSP-1); likewise, we do not detect FSP-1 in our SMC cultures (N. Yang
and J. E. Faber, unpublished data). Rat1 fibroblasts stably
transfected with 1A-,
1B-, 1D-ARs, and NIH
3T3 fibroblasts stably transfected with
2D-ARs, were maintained in Dulbecco's
modified Eagle's medium-high glucose, 10% FBS, 100 units/ml
penicillin, 100 µg/ml streptomycin, and 250 µg/ml G-418.
RNA Preparation.
Frozen tissue was pulverized under liquid
nitrogen, and total RNA from tissues and cell cultures was homogenized
and extracted in acid guanidinium thiocyanate-phenol-chloroform
(Eckhart et al., 1996). Genomic DNA was digested with RQ1 RNase-free
DNase (1 unit per 50 µg of RNA) for 45 min at 37°C, and the absence of contamination was confirmed in each RT-PCR assay by inclusion of a
no-reverse transcriptase tube. RNA concentration was determined spectrophotometrically at A260. Purity
was assessed according to an
A260/A280
ratio of >1.8, and quality was checked by electrophoresis.
Oligonucleotide Primers.
The following oligonucleotides were
used as sense (A) and antisense (B) primers for RT-PCR.
1A-AR: A, 5'-CGAGTCTACGTAGTAGCC-3', B,
5'-GTCTTGGCAGCTTTCTTC-3'; 1B-AR: A,
5'-ATCGTGGCCAAGAGGACC-3', B, 5'-TTTGGCTGCTTTCTTTTC-3';
1D-AR: A, 5'-CGCGTGTACGTGGTCGCAC-3', B,
5'-CTTGGCAGCCTTTTTC-3'; 2D-AR: A,
5'-AGAAACGCTTCACGTTCGTGC-3', B,
5'-TCTGTAAGCAGCACAGCCCGAGC-3'; 2B-AR: A,
5'-CGCCATCGCGTCGGCCATC-3', B, 5'-GAGACCTCTGCAGTGGCTG-3';
2C-AR: A, 5'-CTGGCGGCGGCGGCGGCTGA-3', B,
5'-TCGGGCCGGCGGTAGAAAG-3'; and cyclophilin: A,
5'-ATCCTGAAGCATACAGGTC-3', B, 5'-AGTGAGAACAGAGATTAC-3', which
amplify 204-, 201-, 218-, 271-, 583-, 557-, and 340-bp fragments of the
respective gene transcripts. All primers were synthesized commercially
by Life Technologies. Amplification efficiency was determined in Fig. 1
or as described under Results. Primer pairs amplified
fragments of similar size and location. Sequences of primers for the
2B- and 2C-AR were as
noted by Richman and Regan (1998), and were used for qualitative but
not quantitative RT-PCR, since no product was detected in any fresh or
cultured vascular tissue or cell type.
Construction and Synthesis of Mutant cRNAs.
For quantitative
RT-PCR, 1-AR mutants were made with an
inserted EcoRI site, whereas a BamHI site was
inserted for the 2D-AR mutant. These point
mutations were made at similar locations in the 3rd intracellular loop
sequences with the following sense (A) and antisense (B) primer pairs:
1A-AR: A, 5'-AGACTCAGAGGAATTCACGCTCCGCA-3', B,
5'-TGCGGAGCGTGAATTCCTCTGAGTCT-3'; 1B-AR: A,
5'-TGACCCTGAGAATTCACTCCAAGA-3', B, 5'-TCTTGGAGTGAATTCTCAGGGTCA-3';
1D-AR: A,
5'-GTGGTTCTGAGAATTCACTGTCCGC-3', B,
5'-GCGGACAGTGAATTCTCAGAACCAC-3'; and 2D-AR: A,
5'-TCTGGTTCGGATCCTGCAACAGC-3', B, 5'-GCTGTTGCAGGATCCGAACCAGA-3'. The T3
primer was 5'-AATTAACCCTCACTAAAGGG-3'. The T7 primer was
5'-GTAATACGACTCACTATAGGGC-3'. To generate mutant constructs, cDNA
fragments of 1A-,
1B-, 1D-, and
2D-AR were amplified by RT-PCR of RQ1
RNase-free DNase-treated RNA from tissues abundant in the target RNA
(rat submaxillary gland for 1A-AR, rat liver
for 1B-AR, and rat cerebral cortex for
1D- and 2D-AR) using
the above primer pairs prepared with BamHI and
EcoRI linkers (1A- and
2D-AR) and EcoRI and
SmaI linkers (1B- and
1D-AR). The resultant PCR products were
restricted with these enzymes and subcloned into pBluescript
SK+ vector. Each subclone served as a template to
generate a site-directed mutant fragment by PCR using two pairs of
primers [T3 primer and specific antisense
(1A- and 2D-AR) or
sense (1B- or 1D-AR) oligonucleotide with an EcoRI or BamHI
restriction site in the middle of the primer; T7 primer and the
complementary sequence of the antisense or sense oligonucleotide
primer]. The resultant two PCR products were mixed and re-amplified
with T3/T7 primers. The final PCR product was digested with
XbaI/HindIII, and then cloned into pBluescript
SK+ vector at XbaI/HindIII
sites. All primary clones and mutant clones were confirmed by sequencing.
-AR plasmid constructs, whose products served as competitive
templates for competitive RT-PCR, were linearized with either
HindIII (1A- and
2D-AR) or XbaI
(1B-, 1D-, and
2D-AR), purified with phenol-chloroform
extraction, and precipitated by ethanol. Mutant cDNAs were transcribed
by T3 (1A- and 2D-AR) or T7 (1B- and 1D-AR)
RNA polymerase, in the absence of [32P]CTP, as
described previously (Yang et al., 1999). Transcription products were
digested with RQ1 RNase-free DNase and extracted, precipitated,
dissolved in diethyl pyrocarbonate-treated water, and stored in small
aliquots at 80°C after determination of RNA concentration in
triplicate. Mutant products were differentiated from endogenous PCR
products by restriction enzyme digestion and electrophoresis.
Quantitative Competitive RT-PCR.
Single-tube RT-PCR using
rTth DNA polymerase (Promega, Madison, WI)
(Chiocchia and Smith, 1997) gave superior results when compared with
conventional RT-PCR. Total RNA from tissue or cell culture (amounts
given under Results) was mixed with known amounts of mutant
cRNA competitor, and reverse-transcribed in a 20-µl reaction
containing 200 µM dNTPs, 1 mM MnCl2, 10 mM
Tris-HCl, pH 8.3, 90 mM KCl, 75 µM antisense primer, and 5 units of
rTth DNA polymerase. The reactions were performed
in a GeneAmp 2400 thermocycler using thin-walled MicroAmp reaction
tubes (PerkinElmer, Norwalk, CT) without mineral oil overlay. Reverse
transcription was allowed to proceed for 20 min at 60°C and stopped
on ice. PCR amplification was then carried out in the same tube in a
100-µl volume containing 1.5 mM MgCl2, 10 mM
Tris-HCl, pH 8.3, 100 mM KCl, 0.05% (w/v) Tween 20, 0.75 mM EGTA, 5%
(v/v) glycerol, 0.15 µM sense primer, and 1 µCi of
[32P]dCTP. Initial denaturation at 95°C for
60 s was followed by 35 cycles each of denaturation at 95°C for
15 s and annealing and extension at 62°C for 30 s, with a
final extension at 62°C for 8 min. Forty cycles were performed for
2B- and 2C-AR RT-PCR using both single-tube and standard RT-PCR assays. Thirty cycles were
performed for cyclophilin detection.
2D-AR digested with
BamHI) for 2 h at 37°C to permit differentiation of
target RNA from competitor RNA, followed by electrophoresis on 1.5%
agarose containing 0.5 µg/ml ethidium bromide. Complete digestion of
PCR products arising from amplification of the mutant cRNAs was
confirmed in each assay. Ethidium bromide-stained DNA bands
corresponding to target or competitor products were excised under UV
light, dispersed from the excised bands, and counted (Wallac Microbeta
1450 LSC). RT-PCR for cyclophilin, which like -AR mRNAs, is in low
abundance in many tissues, including arteries (Yang et al., 1999), was
run in parallel in each assay, and excised bands were counted for
correction of intra-assay variability among RT-PCR reactions. The log
of the ratio of the radioactivity of target PCR product to competitive
mutant product was plotted against the log of the competing mutant cRNA
template concentration added to each reaction (Prism, GraphPad
Software, Inc., San Diego, CA). Concentration of target RNA (in
molecules per nanogram of sample RNA) was obtained by interpolation of
the resultant linear regression to the equivalence point, where the
amount of target mRNA present in the sample equals the amount of
competitor RNA (Chiocchia and Smith, 1997). All RT-PCR products, when
first tested against positive control tissues and vascular
cells/tissue, were sequenced for identity.
RNase Protection Assay (RPA).
RPA for -smooth muscle
(-SM)-actin mRNA was performed as described previously (Yang et al.,
1999). For RPA of 2B-AR, the 339-bp
SpeI/KpnI (1245-1584) fragment of the
2B-AR cDNA was cloned into pBluescript
SK+ vector digested with SpeI and
KpnI. A 380-bp cRNA probe was synthesized after
linearization with XbaI. The 179-bp cyclophilin probe
construct was obtained from Ambion (Austin, TX). The identity of
products for these assays have been determined by sequencing (Yang et
al., 1999).
Immunohistochemistry.
Rat thoracic aorta media and
adventitia were examined for -SM-actin. Thoracic aortae were
perfusion-cleared with PBS and fixed at 100 mm Hg with 4%
paraformaldehyde in PBS. Paraffin-embedded, 5-µm sections were
subjected to standard immunohistochemistry using biotinylated
anti-mouse -SM-actin monoclonal antibody (Dako 1A4, 1:50 dilution;
DAKO, Carpinteria, CA) and diaminobenzidine visualization. Adjacent
sections were also stained with Masson's trichrome, nuclear fast red,
and nonimmune mouse IgG.
Membrane Preparation and Radioligand Binding Assay.
For each
binding experiment, purified membrane protein (microsomal preparation)
from 12 aorta media and adventitia (72 rats total) was obtained
according to modifications of Deng et al. (1996). Briefly, frozen
tissue was pulverized in liquid nitrogen, and homogenized in 4°C
buffer [25 mM Tris-HCl, 1 mM EDTA, 2 mM MgCl2,
and 100 mM KCl, pH 7.4, plus the proteinase inhibitors identified above
(see Tissue Collection) and 10 µg/ml leupeptin, trypsin inhibitor, and chymostatin] with a homogenizer (model TH,
Omni, Atlanta, GA) at maximal speed (5 × 10 s). All
procedures below were carried out at 4°C. The homogenate received 3 strokes in a Polytron, treated with 0.6 M KCl for 20 min to remove
major contractile proteins, centrifuged at 1,000g for 10 min, and the postnuclear supernatant centrifuged at 10,000g
for 10 min to sediment mitochondria. The supernatant was centrifuged at
110,000g for 1 h. The final pellet was resuspended in
incubation buffer (50 mM Tris-HCl, 2 mM MgCl2, 1 mM EDTA, pH 7.4, proteinase inhibitors). Purified membrane protein from
SMC and AFB cell cultures was prepared as described above, after
scraping PBS washed cells into homogenization buffer. Crude membrane
protein from Rat1 fibroblasts transfected with -ARs was made as
described by Langin et al. (1989). Crude adult rat kidney membrane
protein was obtained after pulverizing as described above,
homogenization in buffer (50 mM Tris-HCl, 1 mM EDTA, pH 7.5, proteinase
inhibitors), and centrifugation at 2,500g for 10 min. The
supernatant was further centrifuged at 25,000g for 20 min,
and the pellet was resuspended with homogenization buffer,
rehomogenized, and recentrifuged at 25,000g for 20 min. The
final pellet was resuspended in incubation buffer. The concentration of
membrane protein was determined by bicinchoninic acid assay (Pierce,
Rockford, IL).
) in 250-µl total
reaction volumes at 25°C with shaking, and repeated at least three
times on independent membrane preparations. Protein samples assayed
were 20 µg of crude protein for cloned fibroblasts, 50 µg of crude
protein for kidney, and 50 µg of purified protein for media,
adventitia, SMCs, and AFBs. Competition assays used the
1D-AR antagonist BMY 7378 (BMY) (RBI, Natick,
MA) or 1A-AR antagonist KMD 3213 (KMD) (Kissei
Pharm Co, Matsumoto-City, Japan), and a
[3H]prazosin concentration (85 Ci/mmol,
Amersham Pharmacia Biotech, Arlington Heights, IL) (see
Results) equal to its Kd
determined in prior saturation binding assays. Nonspecific binding was
defined by 10 µM phentolamine HCl (RBI) and was <10% for cultured
cell membranes and ~20% for tissue membranes. Data were analyzed
with Prism (GraphPad Software, Inc., San Diego, CA), including ANOVA (p < 0.05) and fitting to one-site versus two-site models.
Protein Synthesis.
Protein synthesis in confluent AFBs that
had been growth-arrested for 24 h in serum-free defined media was
determined during the last 6 h of a subsequent 24-h interval of
continued exposure to norepinephrine or vehicle, using
[35S]methionine incorporation (1000 Ci/mmol,
Amersham) as described previously (Xin et al., 1997). Cell number was
determined by hemocytometry. Protein content was determined by
bicinchoninic acid assay.
| |
Results |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Validation of RT-PCR.
2B- and
2C-AR mRNAs were not detected in aorta media,
adventitia, or cells cultured from these tissues (see below), thus competitive RT-PCR assays for these receptors were not developed. Pilot
studies screened various primer pairs and competitors for the
2D- and the 1-ARs.
Efficiencies were assessed by obtaining the cycle number-amplification
product curve for each target mRNA, permitting identification of the
total amount of RNA and cycle number required to yield a product amount
near the midpoint on the linear portion of each curve. Primer pairs
found to amplify with similar efficiencies (sequences given under
Materials and Methods) were then tested to identify the
amounts of total RNA required, depending on -AR subtype assayed,
that would permit the approximate midpoint of each curve to be obtained
by the same number of cycles for all four -AR mRNAs (35 cycles) and
by 30 cycles for the more abundant cyclophilin. In assays of tissue, 100 ng of RNA was used for 1A-,
1B-, 1D-, and
2D-AR detection. In assays of cultured cells,
100 and 400 ng of RNA were used for 1B-/1D-AR and
1A-/2D-AR,
respectively. Ten nanograms of RNA were used for assay of cyclophilin
in tissue and cells.
1B-AR assay
against 100 ng of SMC RNA is shown in Fig. 1,
left. Thirty-five cycles yielded an 1B-AR product that fell near the midpoint of a
semilog plot of yield against cycle number (Fig. 1, right). Similar
curves were obtained for the other -ARs and cyclophilin. In
competition assays, the midpoint product amount at each competitor
concentration was visualized on ethidium bromide-stained agarose gels,
permitting band excision and [-32P]dCTP
scintillation counting. Examples of resultant competition curves are
shown in Fig. 2 for the
1-AR primer pairs and competitor cRNAs against
RNA from tissues with moderate abundance for each subtype. To determine
assay sensitivities, different known amounts of in vitro transcribed
cRNA for 1D-, 1B-,
1A-, 2D-AR, and cyclophilin [in the context of 100 ng (for
1D-/1B-AR), 400 ng (for 1A-/2D-AR), or
10 ng (for cyclophilin) of yeast carrier tRNA, respectively] were
amplified with 35 (-ARs) or 30 cycles (cyclophilin) against
different ranges of competitor cRNA. Message levels 
0.05 molecule of
target mRNA/ng of total RNA could be consistently detected.
|
|
Aorta Media and Adventitia Express mRNAs for All Three
1-ARs and the 2D-AR.
mRNA transcript
levels were significantly different for each -AR subtype between the
two layers (p < 0.05) (Table
1). Aorta media expressed
transcripts for 1A-,
1B-, 1D-, and
2D-AR of (in fold differences, where
1A-AR = 1) 1, 6, 115, and 1. Aorta adventitia also expressed -ARs, where compared with media, mRNAs were 10- and 21-fold higher for 1A- and
2D-AR, and 15- and 7-fold fold lower for
1B- and 1D-AR.
|
1A- and 2D-AR mRNAs Are Reduced in
Cultured SMCs and AFBs.
Adult rat aorta media is composed entirely
of SMCs [i.e., -SM-actin positive cells (cf. Chen et al., 1995;
Faggin et al., 1999)], so that homogeneous SMC cultures were derived
from it using differential plating. However, cells other than AFBs can occasionally be found in adventitia of rodent arteries (mast cells, macrophages, leukocytes, adipocytes, and several -SM-actin positive cells sometimes present just outside of the external elastic lamina). These cells are larger than AFBs, permitting their exclusion by sieving
after enzymatic dispersion. Also, we have found that non-AFBs make up
less than 1% of rat aorta adventitial cells, as determined by
hemocytometry of dispersed adventitial cells during primary isolation,
and with anti--SM-actin immunohistochemistry. However, with the
exception of activated mast cells in atheromatous plaques that appear
to express 2D-ARs (Handy et al., 1998), there
is no evidence that the few non-AFB cells of the adventitia normally express -ARs.
1D- and
1B-AR mRNAs determined by competitive RT-PCR
were not significantly different between media and SMCs (Table 1). AFBs
also expressed both transcripts, but in levels that were decreased 3- to 5-fold compared with adventitia. In control experiments performed
prior to our competitive RT-PCR studies, we determined whether the
levels of the 1B- and
1D-AR transcripts remain stable with passage using RPAs (Yang et al., 1999). mRNA levels for
1B-AR, 1D-AR, and
cyclophilin were unchanged (p > 0.05, ANOVA) among
pass 3, 4, 5, and 6 for SMCs (expressed as a percentage of pass 4):
1B-AR = 103 ± 22, 100, 141 ± 22, 145 ± 14; 1D-AR = 85 ± 5, 100, 73 ± 10, 93 ± 5; cyclophilin = 110 ± 5, 100, 105 ± 8, 113 ± 5; cyclophilin expression in fresh
media was 111 ± 4% of pass 4 SMCs (n = 6-10 independent RNA samples for each -AR determination, and
n = 3 for cyclophilin).
Unlike 1B-, 1D-AR,
and cyclophilin mRNAs that were maintained at in vivo levels in early
passage-cultured aorta SMCs and AFBs, 1A- and
2D-AR levels in both cell types were reduced
200- to 7000-fold below in vivo levels, as determined by competitive RT-PCR (Table 1, p < 0.05). This marked reduction in
mRNA (and receptors
see below) occurred regardless of aorta SMC cell
culture conditions. These consisted of deriving primary cultures from medial explants instead of enzymatic dispersion, passage number (pass
2-6), level of confluency or serum presence (~70% confluence versus
2 or 11 days after reaching confluence in the presence of 10% FBS or
after 2-3 additional days in serum-free defined medium), plating
surface (glass, plastic, laminin, soluble or fibrillar type I collagen,
elastin, fibronectin, or RGD peptide). The reduction in mRNA was not
prevented by exposure to the following agents and conditions for up to
48 h in serum-free defined medium: 1 µM norepinephrine, 10 nM
angiotensin II, 80 mM KCl, 10 nM dexamethasone, 10 nM
diethylstilbestrol, 25 nM cyclic guanosine monophosphate, 25 nM
forskolin, 10 nM cis-9-retinoic acid, 20 ng/ml fibroblast growth factor 2, 20 ng/ml platelet-derived growth factor-BB, normal tissue oxygen levels (21 mm Hg ambient PO2), and
60-Hz phasic stretch at 20% elongation on Flexercell plates (Flexcell,
Inc., McKeesport, PA). Similar relative levels of
1B- and 1D-AR mRNAs to those in aorta SMCs (Table 1) were found by RPA of SMC cultures derived from adult rat superior mesenteric artery, pulmonary artery, vena cava (data not shown), and magnetically isolated renal afferent arterioles (Salomonsson et al., 2001). (All were passage 4 cells obtained as described under Materials and Methods for aorta,
and 2 days postconfluent plus 2 days in serum-free defined medium; n = 3 for each SMC type). Similarly, the very low
levels of 1A- and
2D-AR mRNAs in aorta SMCs identified by RT-PCR
(Table 1) were undetectable by RPA in these other SMC types, with the
exception of intact thoracic vena cava and pass 4 SMCs from the
pulmonary artery and vena cava, which did express detectable levels of
2D-AR (not shown) in agreement with our
previous studies (Ping and Faber, 1993). RPAs also showed abundant
1A-AR mRNA in intact vena cava (not shown),
possibly because of the mixed SMC and fibroblasts in this vessel.
2B- and 2C-AR Are Not Expressed by
Medial or Adventitial Cells.
Whether using the more sensitive
single-tube method (Fig. 3, left) or the
standard method with separate RT and PCR reactions (data not shown), 40 cycles of PCR with up to 2 µg of RNA did not detect
2B- or 2C-AR mRNAs in
media, adventitia, cultured SMCs, or AFBs from adult rat aorta (Fig. 3,
left). 2B-AR mRNA was also not detected in
aorta tissues and cells by RPAs (Fig. 3, right), but was detected in
intact vena cava and pass 4 SMCs cultured from it (not shown), in
agreement with our previous report using qualitative RT-PCR that used
different primer pairs than herein (Ping and Faber, 1993).
2C-AR expression by RT-PCR was not confirmed
by RPAs because we (Ping and Faber, 1993) and others have not detected
2C-AR mRNA in rat aorta using different primer pairs than herein.
|
-SM-actin in aorta
adventitia after separation from media or when examined intact,
respectively. This is in agreement with the absence of detection of
-SM-actin mRNA in adventitia (Chen et al., 1995). Adventitial
collagen and AFB density are indicated in Fig. 5, A and B.
|
|
1-AR Densities: Validation of Assays Using Cloned
Fibroblasts and Kidney.
Saturation and competition binding assays
were first optimized using cloned fibroblast cultures and rat kidney.
These validation studies were required because of 1) limited
availability of thoracic aorta media and adventitia (media cannot be
separated from adventitia in abdominal aorta), 2) low density of the
total 1-AR population on aorta, and 3) the
need to differentiate all three 1-AR subtypes (based on the above mRNA studies). First, three Rat1 fibroblast cell
lines each stably expressing one of the full-length cloned rat
1-AR subtypes were assayed.
Bmax for 1D-,
1B-, and 1A-ARs in
fibroblasts were 1234 ± 76, 3733 ± 97, and 520 ± 35 fmol/mg of protein, respectively (n = 3 for each cell
line; nonspecific binding averaged 6, 3, and 9%, respectively).
Membranes from each cell line were then combined in a 7:2:1
1D-, 1B-, and
1A-AR receptor proportion to mimic a possible
aorta media distribution suggested by the above (Table 1) abundance of
media mRNA as 1D-AR 
1B-AR > 1A-AR,
labeled with 0.3 nM [3H]prazosin (the average
Kd for
[3H]prazosin obtained from the preceding
saturation binding studies of the three fibroblast lines), and competed
with the 1D-AR antagonist BMY and the
1A-AR antagonist KMD. Reported
pKi values for BMY at cloned rat
receptors range at 1D-AR from 8.2 to 9.1 (average = 8.6), for 1B-AR from 6.2 to
7.0 (average = 6.5), and for 1A-AR from
6.1 to 7.3 (average = 6.5) (Goetz et al., 1995; Piascik et al.,
1995; Suzuki et al., 1997), demonstrating
1D-AR selectivity of 126-fold. KMD exhibits
pKi values for the cloned rat
1A-AR and submandibular gland
1A-AR of 9.3 and 9.8 (average = 9.6), and
showed 56- and 583-fold selectivity versus 1D-
and 1B-ARs, respectively (Shibata et al.,
1995). BMY and KMD competition assays each fit the two-site model best,
and yielded 60 ± 4 and 15 ± 3% as high-affinity sites,
respectively (70 and 10% expected) (Fig.
6).
|
). KMD competition
against 0.25 nM [3H]prazosin gave 45 ± 5% high affinity (taken as 1A-AR) and 55 ± 6% low affinity (taken as 1B- and/or
1D-AR) (Fig. 7, right); since BMY gave 0%
high-affinity sites (taken as 1D-AR), the
low-affinity KMD sites are presumed to be the
1B-AR. These data, and the indirect determination of 1B-AR density that they
yield, agree with binding and functional blood flow studies of rat
kidneys showing that 1A- and
1B-AR predominate, while
1D-AR is essentially undetectable (cf. Blue et
al., 1995; Canessa et al., 1995; Yang et al., 1997; Salomonsson et al.,
2001). These studies validate our assays and confirm the selectivity of
BMY and KMD. The indirect estimate of 1B-AR
density is required because 1B-AR competitive
antagonists have limited selectivity suitable for competition binding
assays when multiple 1-AR subtypes are
suspected of being present (Docherty, 1998).
|
1-ARs (e.g., 72 rats were required
for Fig. 9 data). Therefore, a "two-point" ligand binding assay,
conducted in duplicate, was devised. The assay was first evaluated in
cloned Rat1 fibroblasts and kidney membranes, and estimates of
Bmax and subtype proportions were then
compared with values obtained from the full binding studies. BMY and
KMD were tested at concentrations approximately 10- and 100-fold higher
than their respective Ki values (20 and 3 nM, respectively, for inhibition of
[3H]prazosin binding at its approximate
Kd for vascular tissue/cells (0.1 nM)
and at a 50-fold higher concentration (Fig.
8). Bmax was estimated from the average of specific binding at the two prazosin
concentrations in the absence of BMY or KMD according to the formula:
(specific binding at 0.1 nM [3H]prazosin × 2 + specific binding at 5 nM [3H]prazosin)/2
("control bars", Fig. 8). Thus the derived "two-point" binding
estimate of Bmax for Rat1 membranes
mixed in a 1:1:1 proportion in Fig. 8 (865 ± 54 fmol/mg of
protein; n = 3) was not significantly different from
Bmax determined by full saturation
analysis using 10 concentrations of
[3H]prazosin (1141 ± 43;
n = 3, data not shown). Estimates of the percentage of
1D- and 1A-ARs were
determined as the percentage of 5 nM
[3H]prazosin-specific binding competed away by
200 nM BMY and 30 nM KMD, respectively; the
1B-AR percentage was determined as 100 minus
the combined 1A- and
1D-AR percentages. This yielded the expected
proportion of subtypes in the Rat1 cell membrane mixture (given in key
of Fig. 8). As an additional validation of this method, two-point
binding assays in rat kidney membranes gave a
Bmax (106 ± 4 fmol/mg of
protein; n = 2) that agreed closely with the full
saturation analysis (114 ± 6) shown in Fig. 7. Estimates of
subtype percentages in kidney (1A-AR = 42 ± 9%, 1B-AR = 53 ± 13%,
1D-AR = 5 ± 5%; n = 2) agreed well with full competition analysis (45, 55, and 0%,
respectively) shown in Fig. 7. These validation studies predict that
these two-point saturation and competition assays will yield reasonably
accurate estimations of 1-AR subtype density
in small vascular tissue samples.
|
1-AR Density in Media, Adventitia, and Cultured SMCs
and AFBs.
As determined by the above two-point binding assay
method, both media and adventitia have similar densities of total
1-ARs (Bmax = 101 and 96 fmol/mg, respectively; Fig.
9). Media expressed predominantly
1D-AR, whereas adventitia expressed
predominantly 1A-AR (Fig. 9). The relative
abundance of receptor subtypes and their mRNAs agree for media, but not
for adventitia where mRNA amounts are
1D-AR > 1A-AR
1B-AR (Table 1).
|
1-AR density was approximately 2-fold higher
for SMCs than AFBs (Fig. 10 and Table
1). BMY identified 34% high-affinity (1D-AR)
sites and 66% low-affinity sites in SMCs, and 18% high affinity
(1D-AR) and 82% low affinity in AFBs. Since
culture had little or no effect on 1B-AR (or
1D-AR) mRNA levels, but reduced
1A-AR mRNA in both cell types to almost
undetectable levels (Table 1), the low-affinity KMD sites are likely to
be the 1B-AR. Consistent with this, KMD only
exhibited low-affinity, single-site competition in both cell types
(presumedly competing at 1D- and
1B-AR). These data are interpreted as an
absence of 1A-AR expression. Moreover, in good
agreement with BMY's published selectivity (see above), BMY displayed
pKi values of 8.4 and 8.2 for SMCs and
AFBs at the high-affinity receptor (1D-AR) and
6.5 for both cell types at their low- affinity site (presumed
1B-AR). Notably, the ratio of
1D-AR:1B-AR receptor
density in SMCs (1:2) and AFBs (1:4) is opposite the ratio of mRNAs
(23:1 and 22:1, respectively, Table 1). Total
1-AR densities
(Bmax) in these purified membrane
preparations of SMCs and AFBs are 5- and 3.8-fold higher, respectively,
than in standard crude membrane preparations obtained from SMCs
(Eckhart et al., 1996) and AFBs (data not shown) when assayed at the
same passage number and at 2 days postconfluence plus 2 more days in serum-free defined media. We also found the yield of purified membrane
protein to be 5 times lower than that obtained for crude membrane
fractions from identical numbers of cultured SMCs.
2-AR density was not determined in the present
study because only 2D-AR mRNA was expressed in
media, adventitia, SMCs, and AFBs, and because radioligand binding data
for total 2-AR density have already been
reported for rat aorta (Regan, 1988; Daniel et al., 1991).
|
Stimulation of Adventitial Fibroblasts with Norepinephrine Induces
Proliferation and Protein Synthesis.
Previously, we found that
24-h norepinephrine treatment of Rat1 fibroblasts stably expressing
cloned 1A-, 1B-, or
1D-AR caused similar dose-dependent increases
in protein synthesis. The same effect was obtained for aorta SMCs and
was blocked by prazosin but was unaffected by propranolol or
rauwolscine, thus identifying 1-ARs as being
responsible for this effect (Xin et al., 1997). To determine whether
native -ARs detected herein on AFBs are functionally coupled to
cellular growth pathways, cell proliferation and protein synthesis
([35S]methionine incorporation) were measured
in postconfluent AFBs that had been growth-arrested for 24 h in
serum-free defined medium. Norepinephrine caused increases in protein
synthesis and cell number (Fig. 11).
|
| |
Discussion |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
A major finding of this study was that aorta media and,
unexpectedly, adventitia expressed all three
1-AR subtypes, wherein total
1-AR density was as high in the adventitia,
which is composed mostly of AFBs, as in the media composed of SMCs.
However, the percentage distribution of 1A-,
1B-, and 1D-AR in
media (19, 26, and 55%, respectively) differed from that in adventitia
(44, 37, and 19%, respectively). mRNA distributions qualitatively
followed these receptor distributions (i.e.,
1A-AR < 1B-AR < 1D-AR
for media; 1A-AR > 1B-AR > 1D-AR
for adventitia), but absolute transcript abundances differed greatly
between the two layers. Among 2-ARs, only
2D-AR transcripts were detected, and levels were 21-fold higher in adventitia than in media. Similar to quiescent SMCs, where norepinephrine induced 1-AR-
mediated protein synthesis and hypertrophy (Xin et al., 1997),
norepinephrine induced protein synthesis and proliferation of quiescent
AFBs. This is the first quantitation of 1-AR
subtypes in media and adventitia.
Distribution of mRNA.
Scofield et al. (1995) used competitive
RT-PCR and found that 1A-,
1B-, and 1D-AR mRNA
distribution in intact aorta of adult rat was 67, 15, and 18%,
respectively. A similar distribution was found in intact rat aorta
using RPAs (Xu and Han, 1996). The major source of disagreement between
these and our results likely reflects absence of removal of intima and
separation of media from adventitia in those studies, as well as
methodological differences. Using RPAs (Yang et al., 1999), we also
found all three 1-AR transcripts and the
2D-AR in media and adventitia of adult rat aorta in qualitatively similar proportions to those reported herein. Using competitive RT-PCR, we have also found that the media and adventitia of adult rat carotid artery express the same four -AR subtypes at levels very similar to the aorta values given in Table 1
(J. E. Faber and N. Yang, unpublished data).
used qualitative RT-PCR and reported
2D-, 2B-, and
2C-AR mRNA in explant cultures of SMCs from thoracic aorta of the same size and strain of rats used herein, although confirmation of products by sequencing or Southern
hybridization was not done. In an earlier study using RT-PCR with
primers different from Richman and Regan (1998), we detected
2D- but not 2B- or 2C-AR transcripts in rat thoracic aorta media
and SMCs cultured from this layer by dispersion (Ping and Faber, 1993).
Therefore, in the present study, we used the primers described by
Richman and Regan (1998), but were again unable to detect
2B- or 2C-AR transcripts in aorta media, adventitia, or cells cultured from these
layers, regardless of dispersion, explant, or other conditions. Also,
RPAs failed to detect 2B-AR in media, SMCs,
adventitia, or AFBs (2C-AR RPA was not
tested). In situ hybridization of rabbit aorta (confirmed with RPAs)
detected 2D-AR on medial SMCs and endothelial
cells, but did not detect 2B- or
2C-AR; adventitia was not examined (Handy et
al., 1998). In rat tail artery, in situ hybridization of
2D- and 2C-, but not
2B-AR, was detected (McNeill et al., 1999),
whereas all three 2-ARs were detected by
RT-PCR (Phillips et al., 1997). As in aorta, we have also confirmed the
absence of expression of mRNA for 2B- and
2C-AR by RT-PCR in separated media and
adventitia of the adult rat common carotid artery (J. E. Faber and
N. Yang, unpublished data).
1-AR Density.
Given that fibroblasts are
normally noncontractile and considered "passive" matrix-maintaining
cells, it is surprising that AFBs express
1-ARs in the same total abundance as in the
medial SMC layer. Moreover, 2D-AR mRNA levels
were 21-fold greater in adventitia than in media. We are unaware of
other reports of -ARs on tissue fibroblasts. We confirmed herein
that Rat1, Cos7, and 3T3 fibroblast cell lines do not express
1- or 2-AR
transcripts (not shown). In addition, freshly dispersed rat cardiac
fibroblasts do not express 1-AR mRNAs or
receptors, although they do express 
2-ARs and
angiotensin receptors (Stewart et al., 1994). Our finding that
substantial amounts of mRNAs and receptors for -AR subtypes are
expressed by adventitial cells could explain some of the disagreement in transcript and receptor subtype levels reported previously for
intact arteries.
1-AR
density as fresh media (111 versus 101 fmol/mg), and
1-AR density decreased modestly for cultured
AFBs when compared with fresh adventitia (48 versus 96 fmol/mg). Thus,
our culture conditions largely preserved total 1-AR density. In contrast, expression of
detectable 1A- and 2D-AR binding sites was lost in cultures of
both cell types (and mRNA levels were reduced 200- to 7000-fold), even
when cells were assayed as early as passage 2, and despite 30 different
culture conditions tested (see Results). We are unaware of
other studies that have compared expression of adrenergic mRNAs or
receptor densities in different vascular wall cell types in situ versus in culture.
Function of Multiple -ARs on Vascular SMCs and Adventitial
Fibroblasts.
The predominance of the
1D-AR population in rat aorta media is
consistent with studies showing it mediates contraction of aorta (Goetz
et al., 1995; Piascik et al., 1995; Deng et al., 1996), as well as the
carotid artery and a number of conduit and resistance blood vessels in
the rat (Leech and Faber, 1996; Docherty, 1998). The
1D-AR appears to be the main determinant of
resting sympathetic tone and arterial pressure in rats (Scott et al., 1999), although 1A-AR signals constriction in
certain other blood vessels (e.g., mesenteric resistance vessels) and
regional circulations such as the kidney (Piascik et al., 1996;
Docherty, 1998; Salomonsson et al., 2001). A contractile role for the
1B-AR in rat appears more restricted, having
been implicated for tail and mesenteric arteries (Piascik et al., 1996;
Docherty, 1998) and rat venous vessels (cf. Leech and Faber, 1996). The
functions of the 1A- and
1B-ARs in aorta medial SMCs remain unknown.
2D-AR mRNA in
rat aorta. This subtype signals constriction of arterioles and venules
in rat skeletal muscle (Leech and Faber, 1996). Absence of
2C-AR mRNA in rat aorta vascular cells is
consistent with the lack of an 2C-AR
contraction in some blood vessels. Similarly, we also failed to detect
2B-AR mRNA in aorta layers and cultured SMCs
from other vessels, except rat vena cava and pulmonary artery. Thus the
2C- and 2B-AR may not
contribute to the 2-AR-dependent contraction of rat
aorta (Lues and Schumann, 1984).
As in our previous studies of SMCs, norepinephrine caused proliferation
and protein synthesis in AFBs. In the present study 1A- and 2D-AR mRNA
declined to almost undetectable levels when AFBs were placed into
culture, and no 1A-AR binding sites could be
detected. Since -ARs, at least on SMCs, cause inhibition of proliferation, norepinephrine stimulation of proliferation of AFBs may
be mediated by the 1D- and/or
1B-ARs present on these cells. However,
studies with - and -AR antagonists are required to test this
hypothesis. One or more of the -AR subtypes on AFBs may signal a
trophic action of catecholamines on vascular wall growth and
remodeling. There is growing evidence of a role for adventitial
fibroblasts and their myofibroblast phenotype in a number of vascular
wall diseases, including fibrosis and intimal hyperplasia in
atherosclerosis, graft rejection, and restenosis (Gutterman, 1999).
Migration to intima, matrix regulation, and contracture by AFBs and
myofibroblasts may be modulated by catecholamines through one or more
of the -ARs we detected.
It has been proposed that an AFB population in adult vessels may
represent an embryonic mesenchymal precursor cell that gives rise to
SMCs during vasculogenesis (Faggin et al., 1999). Such a developmental
link between SMCs and AFBs may, together with the presence of dense
adrenergic innervation in the adventitia of most arteries, underlie the
presence of -ARs on these fibroblasts. Additional studies capable of
single cell resolution will be required to determine whether a given
SMC and AFB expresses all four -AR subtypes in the intact media and
adventitia, or if instead variation exists among SMCs and AFB types as
a function of different phenotypes or location of the cells within
their layers. There is evidence that medial SMCs in rats, larger
species, and humans appear to be composed of more than one phenotype
(Bochaton-Piallat et al., 1996; Frid et al., 1999).
In conclusion, all three 1-AR subtype mRNAs
and receptors are expressed by both medial SMCs and adventitial
fibroblasts in rat aorta, and with the same overall
1-AR density in each layer, but with opposite
relative proportions. Like SMCs, stimulation of AFBs with
norepinephrine induces proliferation and protein synthesis. These
findings demonstrate that vascular fibroblasts, like SMCs, are also
capable of expressing abundant -ARs, and underscore the importance
of differentiating between these cell types when quantitating mRNAs or
receptors and drawing inferences from these data about the role of
different -AR subtypes in mediating vascular functions. Moreover,
they raise the possibility that adrenergic nerves, which are abundant
in the adventitia of most vessels, or elevated plasma catecholamines
may regulate fibroblast involvement in vascular wall growth,
remodeling, and disease.
| |
Acknowledgments |
|---|
We thank Kirk McNaughton for histological assistance, X. F. Deng and D. R. Varma (McGill University, Montreal, Quebec, Canada) for advice on membrane preparation for binding assays; D. A. Schwinn (Duke University, Durham, NC) for 1D-
and 1B-AR-transfected fibroblasts; A. S. Goetz and D. L. Saussy (Glaxo-Welcome, Research Triangle Park, NC)
for 1A-AR-transfected fibroblasts; S. M. Lanier (Medical University of South Carolina, Charleston, SC) for
2D-AR transfected fibroblasts stably
expressing adrenoceptors; and Dr. Y. Kurashina (Kissei Pharmaceutical
Co., Matsumoto-City, Japan) for KMD 3213.
| |
Footnotes |
|---|
Accepted for publication March 22, 2001.
Received for publication October 19, 2000.
This research was supported by the National Institutes of Health-National Heart, Lung, and Blood Institute Grant HL-62584.
The studies reported herein have been conducted in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the U.S. National Institutes of Health.
Address correspondence to: Dr. James E. Faber, Department of Cell and Molecular Physiology, 474 MSRB, University of North Carolina, Chapel Hill, NC 27599-7545. E-mail: jefaber{at}med.unc.edu
| |
Abbreviations |
|---|
AR, adrenoreceptor;
SMC, smooth muscle cell;
AFB, adventitial fibroblast;
RT-PCR, reverse transcription-polymerase
chain reaction;
PBS, phosphate-buffered saline;
FBS, fetal bovine
serum;
FSP, fibroblast-specific protein;
bp, base pair;
RPA, RNase
protection assay;
-SM, -smooth muscle;
BMY, BMY 7378;
KMD, KMD
3213.
| |
References |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
1-adrenoceptor subtypes in vitro and in situ.
J Biol Chem
270:
30980-309881D-adrenoceptor subtype in rat myocardium, aorta and other tissues.
Br J Pharmacol
119:
269-276[Medline].1- and 2-adrenoceptors.
Eur J Pharmacol
361:
1-15[Medline].1B-adrenergic receptor gene expression by arterial but not venous vascular smooth muscle.
Am J Physiol
271:
H1599-H16081-adrenoceptors.
Eur J Pharmacol
272:
R5-R6[Medline].1-adrenoceptors on vascular smooth muscle: correlation with the regulation of contraction.
J Pharmacol Exp Ther
290:
452-4632A-adrenoceptors.
Eur J Pharmacol
167:
95-104[Medline].-adrenergic receptor subtypes mediate constriction of arterioles and venules.
Am J Physiol
270:
H710-H722-adrenoceptor agonists.
Naunyn- Schmiedeberg's Arch Pharmacol
328:
160-163[Medline].-Adrenoceptors and vascular regulation: molecular, pharmacologic and clinical correlates.
Pharmacol Ther
72:
215-241[Medline].-adrenoceptor gene expression in arterial and venous smooth muscle.
Am J Physiol
265:
H1501-H15091-adrenergic receptor subtype mRNA in rat tissues and human SK-N-MC neuronal cells: implications for 1-adrenergic receptor subtype classification.
Mol Pharmacol
46:
221-226[Abstract].2-Adrenergic receptors increase cell migration and decrease F-actin labeling in rat aortic smooth muscle cells.
Am J Physiol
274:
C654-C6621C-adrenergic receptor mRNA in adult rat tissues by RNase protection assay and comparison with 1B and 1D.
Biochem Biophys Res Commun
200:
1177-1184[Medline].1-Adrenoceptor subtypes on rat afferent arterioles assessed
by radioligand binding and RT-PCR. Am J Physiol Renal
Physiol 281, in press.-1 adrenergic receptor antagonists in spontaneously hypertensive rats correlates with binding affinity for the 1-d receptor subtype.
FASEB J
13:
A141.1-adrenoceptor subtype proteins in different tissues of neonatal and adult rats.
Can J Physiol Pharmacol
78:
237-243[Medline].1a-adrenoceptor-selective antagonist: characterization using recombinant human 1-adrenoceptors and native tissues.
Mol Pharmacol
48:
250-258[Abstract].1C-adrenergic receptor from cardiac myocytes.
Circ Res
75:
796-8021d-adrenoceptor.
Biochim Biophys Acta
1323:
6-11[Medline].1D-adrenergic receptors and mitogen-activated protein kinase mediate increased protein synthesis by arterial smooth muscle.
Mol Pharmacol
51:
764-775This article has been cited by other articles:
|
|
 |
|
  T. L. Thai and W. J. Arendshorst Mice lacking the ADP ribosyl cyclase CD38 exhibit attenuated renal vasoconstriction to angiotensin II, endothelin-1, and norepinephrine Am J Physiol Renal Physiol, July 1, 2009; 297(1): F169 - F176. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. S. Lyssand, M. C. DeFino, X.-b. Tang, A. L. Hertz, D. B. Feller, J. L. Wacker, M. E. Adams, and C. Hague Blood Pressure Is Regulated by an {alpha}1D-Adrenergic Receptor/Dystrophin Signalosome J. Biol. Chem., July 4, 2008; 283(27): 18792 - 18800. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. C. Vinci, L. Bellik, S. Filippi, F. Ledda, and A. Parenti Trophic effects induced by {alpha}1D-adrenoceptors on endothelial cells are potentiated by hypoxia Am J Physiol Heart Circ Physiol, October 1, 2007; 293(4): H2140 - H2147. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Hosoda, M. Hiroyama, A. Sanbe, J.-i. Birumachi, T. Kitamura, S. Cotecchia, P. C. Simpson, G. Tsujimoto, and A. Tanoue Blockade of both {alpha}1A- and {alpha}1B-adrenergic receptor subtype signaling is required to inhibit neointimal formation in the mouse femoral artery Am J Physiol Heart Circ Physiol, July 1, 2007; 293(1): H514 - H519. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. E. Faber, C. L. Szymeczek, S. Cotecchia, S. A. Thomas, A. Tanoue, G. Tsujimoto, and H. Zhang {alpha}1-Adrenoceptor-dependent vascular hypertrophy and remodeling in murine hypoxic pulmonary hypertension Am J Physiol Heart Circ Physiol, May 1, 2007; 292(5): H2316 - H2323. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. E. Faber, C. L. Szymeczek, S. S. Salvi, and H. Zhang Enhanced {alpha}1-adrenergic trophic activity in pulmonary artery of hypoxic pulmonary hypertensive rats Am J Physiol Heart Circ Physiol, November 1, 2006; 291(5): H2272 - H2281. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Shi, Z.-H. Duan, R. Papay, E. Pluskota, R. J. Gaivin, C. A. de la Motte, E. F. Plow, and D. M. Perez Novel {alpha}1-Adrenergic Receptor Signaling Pathways: Secreted Factors and Interactions with the Extracellular Matrix Mol. Pharmacol., July 1, 2006; 70(1): 129 - 142. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Bernini, F. Franzoni, F. Galetta, A. Moretti, C. Taurino, M. Bardini, G. Santoro, L. Ghiadoni, M. Bernini, and A. Salvetti Carotid Vascular Remodeling in Patients with Pheochromocytoma J. Clin. Endocrinol. Metab., May 1, 2006; 91(5): 1754 - 1760. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Nozik-Grayck, E. J. Whalen, J. S. Stamler, T. J. McMahon, P. Chitano, and C. A. Piantadosi S-nitrosoglutathione inhibits {alpha}1-adrenergic receptor-mediated vasoconstriction and ligand binding in pulmonary artery Am J Physiol Lung Cell Mol Physiol, January 1, 2006; 290(1): L136 - L143. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Chalothorn, H. Zhang, J. A. Clayton, S. A. Thomas, and J. E. Faber Catecholamines augment collateral vessel growth and angiogenesis in hindlimb ischemia Am J Physiol Heart Circ Physiol, August 1, 2005; 289(2): H947 - H959. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. C McGrath, C. Deighan, A. M Briones, M. M. Shafaroudi, M. McBride, J. Adler, S. M Arribas, E. Vila, and C. J Daly New aspects of vascular remodelling: the involvement of all vascular cell types Exp Physiol, July 1, 2005; 90(4): 469 - 475. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Zhang, D. Chalothorn, L. F. Jackson, D. C. Lee, and J. E. Faber Transactivation of Epidermal Growth Factor Receptor Mediates Catecholamine-Induced Growth of Vascular Smooth Muscle Circ. Res., November 12, 2004; 95(10): 989 - 997. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. J. Gonzalez-Cabrera, T. Shi, J. Yun, D. F. McCune, B. R. Rorabaugh, and D. M. Perez Differential Regulation of the Cell Cycle by {alpha}1-Adrenergic Receptor Subtypes Endocrinology, November 1, 2004; 145(11): 5157 - 5167. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Zhang, S. Cotecchia, S. A. Thomas, A. Tanoue, G. Tsujimoto, and J. E. Faber Gene deletion of dopamine {beta}-hydroxylase and {alpha}1-adrenoceptors demonstrates involvement of catecholamines in vascular remodeling Am J Physiol Heart Circ Physiol, November 1, 2004; 287(5): H2106 - H2114. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. E. Wall, J. E. Faber, X. Yang, M. Tsuzaki, and A. J. Banes Norepinephrine-induced calcium signaling and expression of adrenoceptors in avian tendon cells Am J Physiol Cell Physiol, October 1, 2004; 287(4): C912 - C918. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Hiraizumi-Hiraoka, T. Tanaka, H. Yamamoto, F. Suzuki, and I. Muramatsu Identification of {alpha}-1L Adrenoceptor in Rabbit Ear Artery J. Pharmacol. Exp. Ther., September 1, 2004; 310(3): 995 - 1002. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Kobayashi, T. Matsumoto, K. Ooishi, and K. Kamata Differential expression of {alpha}2D-adrenoceptor and eNOS in aortas from early and later stages of diabetes in Goto-Kakizaki rats Am J Physiol Heart Circ Physiol, July 1, 2004; 287(1): H135 - H148. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Chotani, S. Mitra, B. Y. Su, S. Flavahan, A. H. Eid, K. R. Clark, C. R. Montague, H. Paris, D. E. Handy, and N. A. Flavahan Regulation of {alpha}2-adrenoceptors in human vascular smooth muscle cells Am J Physiol Heart Circ Physiol, January 1, 2004; 286(1): H59 - H67. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. A Ross, B. R Rorabaugh, D. Chalothorn, J. Yun, P. J Gonzalez-Cabrera, D. F McCune, M. T Piascik, and D. M Perez The {alpha}1B-adrenergic receptor decreases the inotropic response in the mouse Langendorff heart model Cardiovasc Res, December 1, 2003; 60(3): 598 - 607. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. J. Carrillo, J. Pediani, and G. Milligan Dimers of Class A G Protein-coupled Receptors Function via Agonist-mediated Trans-activation of Associated G Proteins J. Biol. Chem., October 24, 2003; 278(43): 42578 - 42587. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. J. Gonzalez-Cabrera, R. J. Gaivin, J. Yun, S. A. Ross, R. S. Papay, D. F. McCune, B. R. Rorabaugh, and D. M. Perez Genetic Profiling of alpha 1-Adrenergic Receptor Subtypes by Oligonucleotide Microarrays: Coupling to Interleukin-6 Secretion but Differences in STAT3 Phosphorylation and gp-130 Mol. Pharmacol., May 1, 2003; 63(5): 1104 - 1116. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Kalyankrishna and K. U. Malik Norepinephrine-Induced Stimulation of p38 Mitogen-Activated Protein Kinase Is Mediated by Arachidonic Acid Metabolites Generated by Activation of Cytosolic Phospholipase A2 in Vascular Smooth Muscle Cells J. Pharmacol. Exp. Ther., February 1, 2003; 304(2): 761 - 772. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. C. Teeters, C. Erami, H. Zhang, and J. E. Faber Systemic alpha 1A-adrenoceptor antagonist inhibits neointimal growth after balloon injury of rat carotid artery Am J Physiol Heart Circ Physiol, January 1, 2003; 284(1): H385 - H392. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Hague, P. J. Gonzalez-Cabrera, W. B. Jeffries, and P. W. Abel Relationship between alpha 1-Adrenergic Receptor-Induced Contraction and Extracellular Signal-Regulated Kinase Activation in the Bovine Inferior Alveolar Artery J. Pharmacol. Exp. Ther., October 1, 2002; 303(1): 403 - 411. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Erami, H. Zhang, J. G. Ho, D. M. French, and J. E. Faber alpha 1-Adrenoceptor stimulation directly induces growth of vascular wall in vivo Am J Physiol Heart Circ Physiol, October 1, 2002; 283(4): H1577 - H1587. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Zhang, C. S. Facemire, A. J. Banes, and J. E. Faber Different alpha -adrenoceptors mediate migration of vascular smooth muscle cells and adventitial fibroblasts in vitro Am J Physiol Heart Circ Physiol, June 1, 2002; 282(6): H2364 - H2370. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Zhang, C. S. Facemire, A. J. Banes, and J. E. Faber Different alpha -adrenoceptors mediate migration of vascular smooth muscle cells and adventitial fibroblasts in vitro Am J Physiol Heart Circ Physiol, June 1, 2002; 282(6): H2364 - H2370. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Zhang and J. E. Faber Trophic Effect of Norepinephrine on Arterial Intima-Media and Adventitia Is Augmented by Injury and Mediated by Different {alpha}1-Adrenoceptor Subtypes Circ. Res., October 26, 2001; 89(9): 815 - 822. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
competitor (mut) cpm].
