Adenosine A1 Receptors Mediate Mobilization of
Calcium in Human Bronchial Smooth Muscle Cells
Michael F. Ethier and J. Mark Madison
Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts
Adenosine stimulates contraction of airway smooth muscle, but the
mechanism is widely considered indirect, depending on release
of contractile agonists from mast cells and nerves. The goal was to
determine whether adenosine, by itself, directly regulates calcium
signaling in human bronchial smooth muscle cells (HBSMC). Pri-
mary cultures of HBSMC from normal subjects were loaded with
fura 2-AM, and cytosolic calcium concentrations ([Ca2ⴙ]i) were de-
termined ratiometrically by imaging single cells. The nonselec-
tive adenosine receptor agonist, 5ⴕ-N-ethylcarboxamidoadenosine
(NECA), and the adenosine A1 receptor agonist, N6-cyclopentylade-
nosine (CPA), both stimulated rapid, transient increases in [Ca2ⴙ]i.
In contrast, there were no calcium responses to 2-p-(2-carboxyethyl)
phenethylamino-5ⴕ-N-ethylcarboxamido-adenosine (100 nM) or
N6-(3-iodobenzyl)-adenosine-5ⴕ-N-methyluronamide (100 nM), se-
lective agonists at adenosine A2A receptors and adenosine A3 recep-
tors, respectively. Calcium responses to NECA and CPA were in-
hibited by 8-cyclopentyl-1,3-dipropylxanthine, an adenosine A1
receptor antagonist, and by pertussis toxin (PTX). In other experi-
ments, NECA stimulated calcium transients in the absence of extra-
cellular calcium, but not when cells were preincubated in cyclopi-
azonic acid or thapsigargin to empty intracellular calcium stores.
Calcium responses were attenuated by xestospongin C and 2-ami-
noethoxydiphenylborane, inhibitors of inositol trisphosphate (IP3)
receptors, and by U73122, an inhibitor of phospholipase C. It was
concluded that stimulation of adenosine A1 receptors on HBSMC
rapidly mobilizes intracellular calcium stores by a mechanism de-
pendent on PTX-sensitive G proteins, and IP3 signaling. These find-
ings suggest that, in addition to its well-established indirect effects
on HBSMC, adenosine also has direct effects on contractile signaling
pathways.
Keywords: adenosine A1 receptor; calcium; cAMP; human bronchial
smooth muscle; insulin
Adenosine stimulates airway smooth muscle contraction in nor-
mal tissues from humans and animals, and this response is upreg-
ulated in tissues from individuals with asthma and in models
of asthma (1–5). Most evidence suggests that adenosine causes
airway smooth muscle contraction by stimulating mast cells and
nerves to release mediators that secondarily contract the air-
way smooth muscle cell (1, 3, 6–8). However, a few studies have
isolated human airway smooth muscle cells in culture and shown
that adenosine has some direct effects on this cell type and that
adenosine A1, A2A, and A2B receptors are all expressed on normal
human airway smooth muscle cells. For example, A2B receptors
are coupled to the release of cytokines and vascular endothelial
(Received in original form July 26, 2005 and in final form April 28, 2006 )
This study was supported by a grant from the National Institutes of Health
(HL-54143).
Correspondence and requests for reprints should be addressed to Michael F. Ethier,
Department of Medicine, 364 Plantation Street, LRB, Room 370A, University of
Massachusetts Medical School, Worcester, MA 01605. E-mail: Michael.ethier@
umassmed.edu
Am J Respir Cell Mol Biol Vol 35. pp 496–502, 2006
Originally Published in Press as DOI: 10.1165/rcmb.2005-0290OC on May 18, 2006
Internet address: www.atsjournals.org
growth factor and to stimulation of cAMP formation (9–11).
Also, an adenosine A1 receptor agonist inhibited forskolin-
stimulated cAMP production and chronic activation of the aden-
osine A1 receptor has been linked to sensitization of adenylyl
cyclase (9). Interestingly, studies in an allergic rabbit model
showed that activation of adenosine A1 receptors caused contrac-
tion in airway smooth muscle tissue devoid of mast cells and
suggested that A1 receptors are directly coupled to calcium mobi-
lization and contraction (12, 13). Evidence that adenosine A1
receptors directly regulate calcium in airway smooth muscle is
potentially important because individuals with asthma are hyper-
responsive to adenosine (14), and a recent report demonstrated
that this receptor subtype is present in biopsies of asthmatic
human bronchial smooth muscle tissue (15).
Cultured cells are the main, widely used model for studying
isolated airway smooth muscle without interference from other
cell types. Although all four adenosine receptors (A1, A2A, A2B,
and A3) have been shown to stimulate calcium signaling in other
cell types (16), the coupling of cultured airway smooth muscle
cell adenosine receptors to calcium signaling has not been pre-
viously demonstrated. For the current study we hypothesized
that adenosine directly stimulates one or more adenosine recep-
tor subtypes coupled to calcium signaling in airway smooth mus-
cle cells. Using cultured bronchial smooth muscle cells from
normal human donors, the goal was to identify the adenosine
receptor subtype(s), calcium source(s), and signaling pathways
mediating these calcium responses.
MATERIALS AND METHODS
Human Bronchial Smooth Muscle Cell Culture
Human bronchial smooth muscle cells (HBSMC) from three different
human donors were obtained at passage 3 from Cambrex Bioproducts
(Walkersville, MD). These cultured cells were from normal human
donors with no history of asthma. The cells were primary, nontrans-
formed cultures that exhibited fluorescent staining that was positive for
smooth muscle actin and negative for Factor VIII. Cells were seeded
and cultured in smooth muscle growth media (SMGM2; Cambrex) con-
sisting of MCDB-based smooth muscle basal medium (Cambrex) sup-
plemented with 5% fetal bovine serum, 5 ␮g/ml insulin, 2 ng/ml fibro-
blast growth factor, 0.5 ng/ml epidermal growth factor, 50 ␮g/ml
gentamicin, and 50 ng/ml amphotericin B. Cells were maintained at
37⬚C in a humidified atmosphere (5% CO2), fed every 48 h, and passaged
when 80–90% confluent.
Cell Preparation
For calcium imaging, HBSMC (passages 4–6) were seeded in 96-well
special optic plates with thin (0.005-in) bottoms (Corning Inc., Corning,
NY) at a density of 3,500 cells/cm2. Except for experiments described
in Figures 6 and 7, all cells were grown to 50% confluence in SMGM2
then serum-starved by incubation in modified arresting medium (MAM)
consisting of DMEM (2 mM l-glutamine, 100 U/ml penicillin, 100 ␮g/ml
streptomycin) and supplemented with HEPES (25 mM), NaOH
(10 mM), insulin (5.7 ␮g/ml), and transferrin (5 ␮g/ml) (17). After
24 h, cells were washed with a modified Krebs-Ringer-Henseleit buffer
(KRH) (18) containing (in mM): 115 NaCl, 5 KCL, 1 KH2PO4, 1 MgSO4,
25 HEPES, 15 glucose, and 2 CaCl2, and incubated in 2.0 ␮M fura
2-AM for 60 min at room temperature. Then cells were washed twice
with KRH and preincubated in KRH containing adenosine deaminase
Ethier and Madison: Calcium Signaling by Adenosine A1 Receptor 497

(ADA) (1 U/ml) to eliminate any endogenous adenosine released by

the cells (9, 19). After 30 min, agonists, antagonists, and/or inhibitors
were added to culture wells as specified.

Calcium
Fura 2 was excited by computer-controlled 337- and 380-nm ultraviolet
light generated by a nitrogen laser and a nitrogen laser-pumped dye laser,
respectively (Laser Science, Franklin, MA). Each laser alternately fired
pulses (3 ns) at 30 Hz, and the pulses were directed at the cells through
a ⫻40 objective lens (Nikon, Melville, NY). The fluorescent signals
emitted by fura 2 were passed back through the objective to a 455-nm
dichroic mirror, a 475-nm barrier filter (Omega Optics, Brattleboro,
VT), an image intensifier (Xybion Electronic Systems, San Diego, CA),
and captured by a Philips-based frame transfer charge coupled device
(CCD) camera (CCTV, New York, NY). The analog signals from the
camera were digitized with outputs to a personal computer with soft-
ware by Recognition Technology, Inc. (Framingham, MA). As described
previously (20), background from a cell-free region was subtracted before
data acquisition and then an 11 ⫻ 11 pixel area was selected over the cell.
The fluorescence stimulated by alternating pulses of 337- and 380-nm
light were recorded and their ratios plotted. Ratios were converted to
calcium concentrations: [Ca2⫹]i ⫽ Kd · ␤ · (R ⫺ Rmin)/(Rmax ⫺ R),
where Rmax and Rmin are the fluorescence ratios measured in
high and zero calcium, respectively; ␤ is the ratio of fluorescence stimu-
lated by 380 nm light in zero versus high calcium; and Kd (224 nM)
Figure 1. NECA stimulates concentration-dependent calcium responses
is the equilibrium dissociation constant describing calcium binding to
fura 2 (21). in HBSMC. Cells were prepared as described in MATERIALS AND METHODS.
(A ) Representative traces show F340/F380 ratio as a measure of [Ca2⫹]i in
Cyclic AMP single cells exposed to NECA (10 ␮M) and histamine (10 ␮M). (B ) The
maximum increase in [Ca2⫹]i is shown for individual cells exposed to
Confluent HBSMC (passages 4–6) in 24-well plates were serum-starved
different concentrations of NECA (10⫺9–10⫺4 M).
for 24 h by incubation in either MAM or DMEM. Then cultures were
washed with KRH and preincubated in 1,3-dipropyl-8-cyclopentylxan-
thine (DPCPX) (100 nM) or vehicle (KRH) for 30 min. Cells were then
stimulated for 30 s with either vehicle or NECA (50 ␮M), and the
reaction was terminated by addition of 0.1 N HCl containing 0.2% NECA (Figure 1B). Responses of similar magnitude were ob-
Triton X-100. An aliquot of this cell extract was used for determination served in cells from all three donors.
of protein concentration by Bradford assay (Bio-Rad, Hercules, CA) The potent and selective adenosine A1 receptor agonist,
and another aliquot of the same sample was used for determination N6-cyclopentyladenosine (CPA), also caused rapid, transient
of cAMP concentration by competitive immunoassay (R&D Systems, increases in [Ca2⫹]i (Figure 2A). Responses were dose–dependent,
Minneapolis, MN). with maximal responses occurring at 0.1–10 ␮M (Figure 2B)
and, as with NECA, ⵑ 85% of the cells responded to CPA.
Data Analysis Similar responses were observed in cells from all three donors.
Data were expressed as mean ⫾ SEM. Means were compared by Stu- In experiments comparing CPA to other selective adenosine
dent’s t test, and multiple comparisons between means were analyzed receptor agonists, five of six cells responded to CPA (100 nM),
by ANOVA with Newman-Keuls post hoc follow-up testing. For as- and mean [Ca2⫹]i increased from a basal level of 66 ⫾ 12 nM
sessing the effects of different culture media on calcium responses, to a maximum of 501 ⫾ 110 nM, (P ⬍ 0.01, n ⫽ 6) (Figure 3A).
proportions of cells showing any calcium responses to NECA were In contrast, neither 2-p-(2-carboxyethyl)phenethylamino-5⬘-N-
compared by chi-square testing and Fisher’s exact test with Bonferroni
ethylcarboxamidoadenosine (CGS-21680) (100 nM), a selective
correction for multiple comparisons. GraphPad Prism Software (San
agonist at the adenosine A2A receptor, nor N6-(3-iodobenzyl)-
Diego, CA) was used for analyses and P ⬍ 0.05 was considered
significant. adenosine-5⬘-N-methyluronamide (IB-MECA) (100 nM), a se-
lective agonist at the adenosine A3 receptor, stimulated calcium
Materials responses in any cell (Figure 3A).
Fura 2-AM and pluronic F-127 were obtained from Molecular Probes The selective adenosine A1 receptor antagonist, DPCPX, in-
(Eugene, OR). Insulin and transferrin were obtained from Invitrogen hibited calcium responses to both CPA and NECA (Figure 3B).
(Carlsbad, CA). Other reagents were obtained from Sigma (St. Louis, In eight control cells stimulated with 1 ␮M CPA (seven respond-
MO). ers), [Ca2⫹]i increased from 62 ⫾ 10 nM to 783 ⫾ 267 nM (P ⬍
0.01). In contrast, in the presence of DPCPX (100 nM), responses
RESULTS to CPA were attenuated in eight of eight cells tested. Specifically,
when CPA was applied to the cells, [Ca2⫹]i increased from a
For HBSMC, 5⬘-N-ethylcarboxamidoadenosine (NECA), a non- basal value of 55 ⫾ 12 nM to a peak value of only 105 ⫾ 38 nM
selective adenosine receptor agonist that activates all four adeno- (NS, n ⫽ 8). Similarly, when cells were stimulated by NECA
sine receptor subtypes, caused a rapid, transient increase in (10 ␮M) instead of CPA, [Ca2⫹]i increased from 77 ⫾ 8 nM to
[Ca2⫹]i that was similar in magnitude to transients elicited by 1,084 ⫾ 208 nM (P ⬍ 0.001, n ⫽ 10) in the absence of DPCPX,
histamine (Figure 1A). Approximately 85% of cells responded but from 72 ⫾ 8 nM to only 215 ⫾ 89 nM in the presence of
to NECA with a calcium transient. Calcium responses to NECA DPCPX (100 nM) (NS, n ⫽ 9). DPCPX (100 nM) alone had no
were observed at concentrations ranging from 10⫺8–10⫺4 M, and effect on [Ca2⫹]i.
the peak magnitude of the calcium transient was concentra- Pretreatment of HBSMC with pertussis toxin (PTX) (200 ng/ml ⫻
tion dependent, with maximum responses occurring at ⵑ 1 ␮M 24 h), an inhibitor of Gi/Go proteins, attenuated calcium
498 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 35
Figure 2. CPA stimulates concentration-dependent calcium responses.
The adenosine A1 receptor agonist, CPA, was added to HBSMC and
responses recorded. (A ) A representative trace shows the F340/F380 ratio
in a single cell as a measure of [Ca2⫹]i in response to CPA (1 ␮M). (B )
The maximum increase in [Ca2⫹]i is plotted for individual cells exposed
to different concentrations of CPA (10⫺9–10⫺5 M).
transients in response to both CPA and NECA (Figure 3C). In
control cells, [Ca2⫹]i increased from 65 ⫾ 20 nM to 535 ⫾ 98 nM
(P ⬍ 0.001, n ⫽ 7) in response to CPA (1 ␮M). In contrast, in
cells pretreated with PTX, basal [Ca2⫹]i was 64 ⫾ 19 nM and
was only 69 ⫾ 19 nM in response to CPA (NS, n ⫽ 7). Similarly,
in control cells, [Ca2⫹]i increased from 48 ⫾ 9 nM to 715 ⫾
209 nM (P ⬍ 0.001, n ⫽ 5) in response to NECA (10 ␮M), but
in cells pretreated with PTX, basal [Ca2⫹]i was 48 ⫾ 7 nM and
was only 46 ⫾ 5 nM in response to NECA (NS, n ⫽ 5). In
contrast, the peak magnitude of calcium transients in response
to histamine (10 ␮M) was the same with, or without, pretreat-
ment with PTX. Specifically, in response to histamine, [Ca2⫹]i
increased from 63 ⫾ 14 nM to 677 ⫾ 109 nM (n ⫽ 8) and from
97 ⫾ 18 nM to 690 ⫾ 95 nM (n ⫽ 8), without and with pretreat-
ment with PTX, respectively.
To determine the source of calcium supporting these in-
creases in [Ca2⫹]i in response to adenosine receptor agonists,
transients in response to NECA (10 ␮M) were compared and
found to be similar in the presence and absence of extracellular
calcium (Figure 4). In the presence of extracellular calcium,
8 of 10 cells responded (peak increase in [Ca2⫹]i ⫽ 990 ⫾ 237
nM, n ⫽ 10). Similarly, in the absence of extracellular calcium,
six of seven cells responded (peak increase in [Ca2⫹]i ⫽ 755 ⫾
173 nM, n ⫽ 7). In other experiments, in the presence of extra-
cellular calcium, HBSMC were treated with cyclopiazonic acid
(30 ␮M) or thapsigargin (0.3 ␮M) for 20 min to deplete intracellu-
lar calcium stores by inhibiting sarco-endoplasmic reticulum
calcium ATPase (SERCA) (20, 22). No responses to NECA
were observed after pretreatment with either cyclopiazonic acid
(n ⫽ 6) or thapsigargin (n ⫽ 6) (Figure 4).
2006
Figure 3. Adenosine A1 receptor signaling stimulates calcium responses
in HBSMC. (A ) Agonists (100 nM) selective for adenosine receptor
subtypes were added to HBSMC and calcium responses recorded. (B )
CPA (1 ␮M) or NECA (10 ␮M) was added to HBSMC in the absence
or presence of DPCPX (100 nM), an adenosine A1 receptor antagonist.
(C ) CPA (1 ␮M), NECA (10 ␮M), or histamine (10 ␮M) was added to
HBSMC with or without pretreatment with PTX (200 ng/ml ⫻ 24 h).
Data are mean ⫾ SEM for basal (open bars) and maximal [Ca2⫹]i (solid
bars) (n ⫽ 5–9). *P ⬍ 0.05 compared with vehicle, †P ⬍ 0.05 compared
with agonist without DPCPX (B ) or PTX (C ).
In the presence of 2-aminoethoxydiphenylborane (2-APB)
and xestospongin C, cell-permeable inhibitors of the inositol
trisphosphate (IP3) receptor (IP3R), calcium responses to NECA
were inhibited (Figure 5). In five control cells, [Ca2⫹]i increased
by 1,019 ⫾ 335 nM in response to NECA (10 ␮M), but in the
presence of xestospongin C the maximum increase in [Ca2⫹]i
was significantly less (312 ⫾ 197 nM, P ⬍ 0.05, n ⫽ 5). Similarly,
in the presence of 2-APB, [Ca2⫹]i did not increase in response
to NECA (n ⫽ 4). The presence of a phospholipase C (PLC)
inhibitor, 1-[6-[((17␤)-3-methoxyestra-1,3,5[10]-trien-17-yl)ami-
no]hexyl]-1H-pyrrole-2,5-dione (U73122), also significantly in-
hibited calcium responses to NECA (Figure 5). In the presence
of U73122, [Ca2⫹]i was increased by only 4 ⫾ 4 nM (NS, n ⫽ 5)
in response to NECA.
At the outset of these projects it appeared that calcium re-
sponses to NECA were more likely to occur when cells were
preincubated for 24 h in a serum-free modified arresting medium
Ethier and Madison: Calcium Signaling by Adenosine A1 Receptor
Figure 4. Calcium transients in response to NECA depend on mobiliza-
tion of calcium from intracellular stores. NECA (10 ␮M) was added to
HBSMC in the presence (I) or absence (II) of extracellular calcium, or
after pretreatment with cyclopiazonic acid (30 ␮M) (III) or thapsigargin
(0.3 ␮M) (IV) in the presence of extracellular calcium. (A ) Representative
traces plotting the F340/F380 ratio show responses to NECA. (B ) [Ca2⫹]i
before NECA (basal, open bars) and maximum (solid bars) and plateau
(hatched bars) [Ca2⫹]i after NECA are shown. Data are mean ⫾ SEM for
6–10 cells. *P ⬍ 0.05 compared with basal, †P ⬍ 0.05 compared with
maximum responses to NECA in the presence of extracellular calcium.
(MAM) and exposed to ADA for 30 min before experiments.
For this reason, experiments described above were performed
under these culture conditions. We then performed additional
experiments to determine what components of these conditions
were important for eliciting responses to NECA. To assess
whether the presence of ADA was important for responses to
NECA, we exposed cells to ADA (1 U/ml) for 30 min or 24 h
and found that, for both incubation times, the proportion of
responding cells was similar to cells not exposed to ADA. After
Figure 5. Stimulation of calcium responses by NECA is inhibited by
U73122, xestospongin C, and 2-APB. Cells were stimulated with NECA
(10 ␮M) in the absence or presence of U73122 (5 ␮M ⫻ 60 min),
xestospongin C (xest C) (20 ␮M ⫻ 60 min), or 2-APB (10 ␮M ⫻
30 min). Data are mean ⫾ SEM (n ⫽ 4–5 cells) for basal (open bars)
and maximum (solid bars) [Ca2⫹]i. *P ⬍ 0.05 compared with basal,
†
P ⬍ 0.05 compared with response to NECA in controls.
499
30 min in ADA or vehicle, calcium responses to NECA (10 ␮M)
were observed in 10 of 11 and 6 of 8 cells, respectively. Similarly,
after 24 h in ADA or vehicle, 6 of 8 and 7 of 8 cells responded,
respectively. Adding ADA alone had no effect on [Ca2⫹]i. Based
on these results, it appeared that addition of ADA did not play
a role in optimizing responses to NECA.
Because MAM is composed of DMEM supplemented with
insulin, transferrin, and HEPES, in separate experiments we
investigated which component(s) in MAM favored calcium re-
sponses to NECA. Calcium responses to NECA (10 ␮M) were
assessed after 24 h incubation in media containing various combi-
nations of MAM components (Figure 6). There was a broad
range of calcium responses in all five media tested (Figure 6A),
but the fraction of cells responding to NECA with increased
[Ca2⫹]i was significantly greater when cells were preincubated
in media containing insulin (i.e., MAM or DMEM plus insulin)
(P ⬍ 0.005) (Figure 6B). In all of the media tested, and for
responding and nonresponding cells, there were no differences
in basal [Ca2⫹]i and no morphologic differences by phase contrast
microscopy. In other experiments we were unable to increase
the proportion of cells responding to NECA (10 ␮M) in DMEM
(no insulin) by including ADA (1 U/ml) in the DMEM. After
24 h in DMEM with or without ADA, 6 of 12 and 5 of 12 cells
responded, respectively.
In many cell types, adenosine A1 receptors are negatively cou-
pled to adenylyl cyclase via Gi and A2 receptors are coupled to
Figure 6. Calcium responses to NECA are modulated by insulin. HBSMC
were seeded and grown to 50% confluence in SMGM2 as described in
MATERIALS AND METHODS. Medium was replaced with MAM (which con-
tains insulin), DMEM, DMEM with HEPES (25 mM), DMEM with insulin
(5.7 ␮g/ml), or DMEM with transferrin (5 ␮g/ml) for 24 h, and then
cells were washed and loaded with fura 2-AM. The adenosine receptor
agonist, NECA (10 ␮M), was then added to cells and calcium responses
recorded. (A ) The peak increase in [Ca2⫹]i is plotted for individual cells
stimulated with NECA (open diamonds). Filled diamonds are the mean ⫾
SEM for each group (n ⫽ 12–33). (B ) The proportion of cells responding
to NECA was significantly greater in cells incubated in media containing
insulin (*significantly different than media not containing insulin, P ⬍
0.005).
500 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 35
stimulation of adenylyl cyclase via Gs (16). The nonselective adeno-
sine receptor agonist, NECA, activates both receptor subtypes. For
cells preincubated 24 h in MAM (contains insulin), the overall
effect of NECA (50 ␮M) was a cAMP increase from 3.7 ⫾ 0.2 to
24.0 ⫾ 1.5 pmoles/mg protein (P ⬍ 0.001, n ⫽ 8–9) (Figure 7A).
After preincubation for 30 min in DPCPX (100 nM), a selective
adenosine A1 receptor antagonist, stimulation of cAMP levels
by NECA was increased to 31.0 ⫾ 2.9 pmoles/mg protein, a
value significantly greater than that achieved in the absence of
DPCPX (P ⬍ 0.04, n ⫽ 9). The finding is consistent with the
presence of A1 receptors on these cells and suggests that when
the nonselective agonist, NECA, is applied by itself, there is a
mixed effect on cAMP levels because NECA activates both A1
and A2 receptors. In contrast, for cells preincubated 24 h in
DMEM (no insulin), NECA still increased cAMP from 4.7 ⫾
0.4 to 28.0 ⫾ 2.4 pmoles/mg protein, but this response was not
enhanced by A1 receptor antagonism with DPCPX (27.9 ⫾
3.7 pmoles/mg protein, NS) (Figure 7B).
DISCUSSION
The main finding of this study is that adenosine receptor agonists
stimulate large, transient increases in [Ca2⫹]i (calcium transients)
in normal HBSMC. These calcium responses to adenosine ago-
nists are mediated by adenosine A1 receptors that are coupled to
PTX-sensitive G proteins and IP3 signaling pathways to mobilize
calcium from intracellular stores. These findings are novel be-
cause calcium transients in direct response to adenosine receptor
agonists have not been demonstrated previously in cultures of
human airway smooth muscle cells. Because calcium is an impor-
tant regulator of cell contraction, these findings suggest the possi-
bility that, in addition to the well-established, indirect effects
that it has on airway smooth muscle function, adenosine may
also have direct effects that regulate bronchomotor tone in the
airways.
In the current study, for HBSMC, calcium transients in re-
sponse to adenosine were mediated by the adenosine A1 recep-
Figure 7. Stimulation of
cAMP by NECA is increased
by DPCPX. HBSMC were
serum-starved for 24 h by
incubation in either (A )
MAM (insulin-containing
media with frequent cal-
cium responses, see Figure
6) or (B) DMEM (non–
insulin-containing media
with infrequent calcium re-
sponses, see Figure 6). Cul-
tures were washed, incu-
bated for 30 min in vehicle
or DPCPX (100 nM), and
either vehicle or NECA
(50 ␮M) was added for 30 s.
Cyclic AMP was measured
as described in MATERIALS
AND METHODS. Data are
mean ⫾ SEM. SEM for
vehicle controls were
⬍ 1 pmole/mg and, there-
fore, not visible on figure.
*P ⬍ 0.001 compared with
vehicle control, †P ⬍ 0.05
compared with NECA
alone (n ⫽ 8–9).
2006
tor. Several findings support this. First, calcium responses were
elicited by CPA, a selective adenosine A1 agonist, and NECA,
which activates all four of the known adenosine receptor sub-
types (A1, A2A, A2B, and A3). Responses to both of these agonists
were concentration dependent and, consistent with responses
mediated by adenosine A1 receptors, were detected at concentra-
tions as low as 10 nM (16, 23). Second, additional evidence that
adenosine A1 receptors mediated the responses was the finding
that agonists selective for adenosine A2A and A3 receptors did not
stimulate calcium transients in these cells. The lack of response to
an A3 agonist is significant because a study of rat tracheal smooth
muscle cells previously showed that A3 receptor activation en-
hances calcium transients in response to the purine nucleotide,
ATP (24). Third, also consistent with the presence of A1 recep-
tors on these cells, the nonselective adenosine agonist, NECA,
increased cAMP levels more when an A1 receptor antagonist
was present.
Although highly selective agonists for the adenosine A2B re-
ceptor are not available (16, 25), three separate findings all
support the conclusion that the adenosine A1 receptor, not A2B,
is the receptor subtype coupled to calcium transients in HBSMC.
First, the low concentrations (100 nM) of adenosine agonists
selective for the A1, A2A, and A3 receptors in this study do not
significantly activate A2B receptors (11, 16). Second, a low con-
centration (100 nM) of the selective adenosine A1 receptor antag-
onist, DPCPX, inhibited calcium responses to both CPA and
NECA. Finally, adenosine A1 receptors, but not adenosine A2B
receptors, are known to be coupled to PTX-sensitive G proteins
in other cell types (13, 16, 25–27). In the current study, NECA-
and CPA-stimulated calcium transients were inhibited by PTX.
This finding was not a nonspecific effect of PTX on calcium
signaling in general because mobilization of calcium by a differ-
ent agonist, histamine, was unaffected by the same preincubation
in PTX. Therefore, in aggregate, all these findings are most
consistent with adenosine A1 receptors, not adenosine A2B recep-
tors, mediating the generation of calcium transients in HBSMC.
To begin to assess the mechanism underlying adenosine A1
receptor–stimulated increases in [Ca2⫹]i, the source of the cal-
cium ions supporting the transient was evaluated. Because
NECA stimulated calcium transients in calcium-free media, it
was concluded that intracellular stores were the main source of
calcium for transients. Consistent with this, depleting SR calcium
by preincubating the cells in thapsigargin or cyclopiazonic acid,
both inhibitors of SERCA, abolished subsequent calcium tran-
sients in response to NECA. Therefore, adenosine A1 receptors
mobilize calcium from the SR. Because responses to adenosine
agonists also were attenuated by an inhibitor of PLC and by
inhibitors of IP3R, it was further concluded that PLC activation,
IP3 generation, and calcium release through IP3R are the signal-
ing events coupling adenosine A1 receptor activation to the re-
lease of calcium ions from the SR. That adenosine A1 receptors
could couple to PLC through a PTX-sensitive G protein is possi-
ble because studies in a variety of cells and tissues have demon-
strated calcium mobilization stimulated by adenosine A1 recep-
tor coupling to Gi (13, 16, 26, 27), probably involving the
activation of PLC-␤ by G protein ␤␥ subunits (26–29). It is
acknowledged, however, that our findings do not exclude an
alternative possibility that A1 receptor activation stimulates the
release of an unidentified mediator from HBSMC that second-
arily stimulates PLC activation and calcium mobilization.
Finding that adenosine A1 receptors are coupled to calcium
in HBSMC suggests that this receptor subtype could play a
role in regulating smooth muscle cell functions in the airways.
Previous studies reporting direct effects of adenosine on cell
signaling in human airway smooth muscle have identified A2B
as the predominant functionally coupled adenosine receptor
Ethier and Madison: Calcium Signaling by Adenosine A1 Receptor
subtype (9–11). The adenosine A2B receptor is a low affinity
receptor that is coupled to Gs and, possibly, Gq, but not to Gi
or Go (16, 25) and activation of the A2B receptor in human airway
smooth muscle stimulates release of cytokines and vascular endo-
thelial growth factor (10, 11) as well as cAMP accumulation
(9). Consistent with there being both A1 and A2B receptors on
HBSMC, we found that the non-selective adenosine receptor
agonist, NECA, increased cAMP levels more when a specific
adenosine A1 antagonist was present in the media. Although
this finding constitutes additional evidence that A1 receptors are
present on HBSMC, it is acknowledged that our data do not
necessarily indicate that A1 receptors are coupled to Gi and
adenylyl cyclase directly because cAMP levels could have been
regulated by other mechanisms, including activation of phospho-
diesterase and calcium signaling. However, another study of
human airway smooth muscle cells has shown that, in the pres-
ence of a phosphodiesterase inhibitor, forskolin-stimulated
cAMP accumulation was inhibited by the adenosine A1 agonist,
CPA (9). In aggregate, our findings with cAMP do not define
the mechanism of A1 coupling but are consistent with prior
evidence showing that both adenosine A1 and A2B receptors are
expressed by human airway smooth muscle cells (9–11).
Several reasons may explain why calcium transients in re-
sponse to adenosine were observed in the present study, but not
in previous studies of airway smooth muscle cells from human
and rat trachea (8, 22, 30). Perhaps most important, it is possible
that the bronchial origin of the cells used in the present study
was an important difference compared with earlier studies that
cultured tracheal cells (9, 24, 30). In support of this possibility,
in an allergic rabbit model, smooth muscle from the peripheral
airways contracted more in response to adenosine than smooth
muscle from the trachea (31). Second, a feature of the current
study was that changes in [Ca2⫹]i were measured in single cells
rather than in a population of cells (24, 30). Studies that em-
ployed simultaneous measurement of [Ca2⫹]i in multiple cells
might not have detected significant changes if calcium responses
were occurring in a subpopulation of cells. Third, our inclusion
of insulin in the culture medium 24 h before experiments was
important for establishing optimal conditions for A1 receptor
coupling to both calcium signaling and inhibition of cAMP. How-
ever, because a subpopulation of cells responded to NECA even
in media without insulin, the presence of insulin does not appear
to be an absolute requirement.
Adenosine A1 receptor activation has been linked to insulin
sensitivity in muscle tissue and adipocytes (32, 33) but we are not
aware of prior studies showing that insulin treatment enhances
responses that are coupled to A1 receptors. The reason adeno-
sine-stimulated calcium responses are more frequent among
HBSMC incubated in media containing insulin is not known.
Our data do suggest the effect of insulin-containing media also
extends to other cellular functions coupled to A1 receptors, spe-
cifically A1 receptor inhibition of cAMP. Because media con-
taining insulin optimized both calcium responses and inhibition
of cAMP, we speculate that a common, proximal point in these
two different signaling pathways is being modulated by insulin,
for example at the adenosine receptor or G protein levels. How-
ever, it is acknowledged that our data do not address directly
the mechanism underlying effects of these media on A1 receptor
responses and do not exclude effects downstream of G proteins.
We also considered the possibility that adenosine release into
the culture medium might be regulated by the presence of insulin
and that this could contribute to our findings. However, against
this, incubating cultures in different media (with and without
insulin) for up to 24 h in ADA did not affect calcium responses
to A1 receptor stimulation.
501
In vivo and in vitro studies in normal humans and animals
have shown airway smooth muscle contraction or bronchocon-
striction in response to adenosine (1–5). However, contractile
responses to adenosine in mixed cell-type preparations are
widely believed to be indirect and dependent on mediator release
from mast cells and nerves (1–8). Our findings suggest that cau-
tion is warranted when making an assumption that adenosine-
stimulated airway smooth muscle contraction is always indirect
and mediated only by adenosine acting first on cells other than
smooth muscle. Although our cells are derived from normal
subjects, we speculate that our findings may be relevant in asthma
because it is well-established that responses to adenosine are
increased in asthma (1, 14), and a recent study has identified
adenosine A1 receptors in the airway smooth muscle of subjects
with asthma (15).
In summary, for single HBSMC, adenosine A1 receptors are
coupled to calcium signaling, a second messenger pathway im-
portant to the regulation of bronchomotor tone. The calcium
responses are mediated by adenosine A1 receptors coupled to
PTX-sensitive G proteins and depend on the activation of PLC
and the mobilization of calcium from the SR. These findings
indicate that adenosine can directly regulate calcium mobiliza-
tion in airway smooth muscle cells and that HBSMC are a poten-
tially useful model for studying these direct effects of adenosine.
Conflict of Interest Statement : Neither author has a financial relationship with a
commercial entity that has an interest in the subject of this manuscript.
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