Br. J. Pharmacol. (1993), 108, 85-92 '.

" Macmillan Press Ltd, 1993

Adenosine A1-receptor stimulated increases in intracellular

calcium in the smooth muscle cell line, DDT1MF-2
'John M. Dickenson & Stephen J. Hill
Department of Physiology and Pharmacology, Medical School, Queen's Medical Centre, Nottingham, NG7 2UH
1 The effect of of adenosine receptor agonists on intracellular free calcium concentration
a range
vas deferens smooth muscle cell line DDTMF-2.
([Ca2+]j) has been studied in the hamster
2 Adenosine receptor agonists elicited a rapid and maintained increase in [Ca2+], in fura-2 loaded
DDTIMF-2 cells. The initial rise could be maintained in the absence of extracellular calcium, whereas
the maintained or plateau phase was dependent upon the presence of extracellular calcium and appeared
to be associated with calcium influx. The rank order of agonist potencies was N6-
cyclopentyladenosine > 5'-N-ethylcarboxamidoadenosine > 2-chloroadenosine > adenosine.
3 The response to 2-chloroadenosine was antagonized by the antagonists 8-cyclopentyl-1,3-
dipropylxanthine (DPCPX, KD 0.14nM) and 8-phenyltheophylline (KD 112 nM).
4 Pretreatment with the 5-lipoxygenase inhibitor AA861 (20 jLM) produced only a small (14 ± 2%)
inhibition of the [Ca2+]i response elicted by N6-cyclopentyladenosine (300 nM), in nominally Ca2'-free
buffer containing 0.1 mM EGTA. The cyclo-oxygenase inhibitor, indomethacin (2 pM) was without
effect.
5 The Ca2"-influx associated with the plateau phase required the continued presence of agonist on the
receptor. The antagonist DPCPX (100 nM) attenuated the rise in [Ca2+]1 observed when extracellular
Ca was re-applied after the cells had been stimulated with N6-cyclopentyladenosine (CPA;300 nM) in
experiments initiated in nominally Ca2+-free buffer.
6 Pretreatment with pertussis toxin (200 ng ml-' for 4 h) inhibited the CPA (100 nM) stimulated
intracellular Ca2+ release and Ca2" influx but was without effect on the response to histamine (100 fiM).
7 These data suggest that adenosine Al-receptor activation in DDT1MF-2 cells stimulates release of
Ca2+ from intracellular stores and influx of extracellular Ca2+ through Ca2+ entry pathways in the
plasma membrane which required the continued presence of agonist on the receptor.
Keywords: Adenosine; A,-adenosine receptors; intracellular calcium; DDTMF-2 cells; smooth muscle

Introduction
Adenosine receptors are widely distributed in mammalian
tissue and have been divided into two major subtypes Al and
A2 (for reviews see Stiles, 1986; 1992). Adenosine A,-
receptors are negatively coupled to adenylate cyclase via a
pertussis toxin-sensitive Gi guanosine 5'-triphosphate (GTP)-
binding protein, whereas adenosine A2-receptors are
positively coupled to adenylate cyclase via a G, protein
(Stiles, 1992). In addition, Al-receptors are now known to
regulate a variety of different effector systems. These include:
(1) opening of potassium channels through pertussis toxin-
sensitive Gi proteins in cardiac tissue (Kurachi et al., 1986),
(2) inhibition of Ca2" channel opening in neurones (Olsson &
Pearson, 1990), (3) activation of guanylate cyclase in smooth
muscle cells (Kurtz, 1987), (4) inhibition of phospholipase A2
activity in hamster brown adipocytes (Schimmel & Elliott,
1988), (5) stimulation of the glucose transporter (Londos et
al., 1981) and (6) the modulation of agonist-stimulated
inositol phospholipid hydrolysis (Linden & Delahunty, 1989;
Hill & Kendall, 1989).
The intracellular cross-talk between adenosine-receptor
stimulation and agonist-induced inositol phospholipid hyd-
rolysis has been widely studied in the central nervous system
(CNS). For example, the activation of adenosine Al receptors
in guinea-pig cerebral cortical slices (Hollingsworth et al.,
1986; Hill & Kendall, 1987) augments the histamine Hl-
receptor induced accumulation of inositol phosphates,
whereas in the cerebral cortex of mouse (Kendall & Hill,
1988) and man (Kendall & Firth, 1990) the histamine res-
I Author for correspondence.
ponse is attenuated. In all three species, adenosine-receptor
activation failed to stimulate inositol phospholipid hydrolysis
on its own. Intracellular cross-talk between adenosine-
receptors and agonists coupled to inositol phospholipid hyd-
rolysis has also been demonstrated in cells grown in culture.
Inhibitions of agonist-induced inositol phospholipid hyd-
rolysis have been described in the rat pituitary cell line GH3
(Delahunty et al., 1988) and the human astrocytoma cell line
1321NI (Nakahata et al., 1991), whereas in FRTL-5 thyroid
cells adenosine-receptor agonists enhanced a,-adrenoceptor-
stimulated inositol phospholipid hydrolysis (Okajima et al.,
1989). However, in addition to these modulatory effects there
are several accounts of adenosine-receptor agonists
stimulating inositol phospholipid directly. The adenosine A,-
receptor mediates the inositol phospholipid hydrolysis res-
ponses found in guinea-pig myometrium (Schiemann et al.,
1991) and the rabbit cortical collecting tubule cell line
RCCT-28A (Arend et al., 1989). In contrast, in the rat
tumour-derived mast cell line RBL-2H3 the order of agonist
potencies stimulating inositol phospholipid hydrolysis was
characteristic of the adenosine A2 receptor (Ali et al., 1990).
Interestingly, the adenosine Al-receptor stimulated inositol
phospholipid hydrolysis in the guinea-pig myometrium is
completely blocked by cyclo-oxygenase inhibitors suggesting
that inositol phospholipid hydrolysis is secondary to syn-
thesis of prostaglandins from arachidonic acid. Measure-
ments of adenosine-receptor induced changes in intracellular
free Ca2" concentration ([Ca2']i) have also been obtained in
the renal epithelial cell line LLC-PK, (Weinberg et al., 1989),
rat cultured mesangial cells (Olivera et al., 1992), cortical
collecting tubule cells (Arend et al., 1988), RBL-2H3 cells
(Hide & Beavan, 1991) and human tracheal epithelial cells
86 J.M. DICKENSON & S.J. HILL
(Galietta et al., 1992).
The DDTIMF-2 smooth muscle cell line, derived from
hamster vas deferens (Norris et al., 1974) expresses both
adenosine Al- and A2-receptors that are respectively coupled
negatively and positively to adenylate cyclase (Gerwins et al.,
1990; Ramkumar et al., 1990). Our previous studies (White et
al., 1992) and those of Gerwins & Fredholm (1991) have also
shown that adenosine Al-receptor activation in DDTMF-2
cells results in inositol phospholipid hydrolysis. In contrast,
Schachter et al. (1992) recently showed that in DDT1MF-2
cells adenosine Al-receptor activation potentiated the inositol
phospholipid response elicited by noradrenaline, but in the
absence of noradrenaline caused only a minor stimulation of
phospholipase C, which was abolished in the absence of
extracellular calcium.
In DDTMF-2 cells we have previously shown that a single
concentration of 2-chloroadenosine (10 jM) elicited a
significant rise in [Ca2J]i in nominally Ca2"-free buffer con-
taining 0.1 mM EGTA (White et al., 1992). In this present
paper we have examined the pharmacological characteristics
of the adenosine-receptor mediated increases in [Ca2+]i. Fur-
thermore, we clearly demonstrate that adenosine-receptor
stimulated increases in [Ca2+]i, measured in nominally Ca2+-
free buffer containing 0.1 mM EGTA, can occur in the
presence of 5-lipoxygenase and cyclo-oxygenase inhibitors.
These data suggest that in DDTMF-2 cells the adenosine-
receptor mediated increase in [Ca2+]i is a consequence of
direct receptor coupling to phospholipase C rather than
being secondary to the products of arachidonic acid
metabolism.
Methods
Cell culture
The hamster vas deferens smooth muscle cell (DDTIMF-2)
was obtained from the European Collection of Animal Cell
Cultures (Porton Down, Salisbury, U.K.). DDTMF-2 cells
were cultured at 37°C in a humidified air/CO2 (90:10) atmo-
sphere in 75cm2 flasks (Costar). The growth medium was
Dulbecco's modified Eagle's medium supplemented with
2mM-L-glutamine and 10% (v/v) foetal calf serum (FCS).
Cells were passaged twice a week (1/6 split ratio) by vigorous
shaking of the flask and placed into 75 cm2 flasks and fed
with fresh growth medium every 48 h. Cells for [Ca2+]i deter-
minations were grown on 24 mm x 10 mm glass coverslips in
90 mm petri dishes. All experiments were performed on
confluent monolayers (passages 4-17, numbers assigned after
receiving the cell line).
Measurement of intracellular free calcium
Intracellular free calcium was measured by loading confluent
cell monolayers with the calcium-sensitive fluorescent dye
fura-2. Individual coverslips were placed in 35 mm petri
dishes with 1 ml of physiological buffer (composition, mM:
NaCI 145, glucose 10, KCI 5, MgSO4 1, HEPES 10, CaCl2 2,
pH 7.45) containing 10% FCS (v/v), 3 jIM fura-2/AM and
incubated for 30 min at 37'C. After this 'loading' period the
fura-2 containing buffer was replaced with fresh buffer, that
was free of fura-2 and FCS but contained 0.1% bovine
serum albumin, and left at 37°C for a further 15 min. Loaded
coverslips were then mounted in a specially designed holder
which enabled the coverslip to be positioned across the
diagonal of a polymethacrylate cuvette. Each cuvette con-
tained 2.9 ml of physiological buffer (drugs were added to the
cuvettes in 100 ftl aliquots) and fluorescent measurements
were made at 37°C by use of a Perkin Elmer LS 50 spect-
rometer. The excitation wavelengths were 340 and 380 nm,
with emission at 500 nm. The slit-widths were set at 10 nm
for both the excitation and emission wavelengths and the
time taken to switch between 340 and 380 nm was 0.8 s.
Intracellular free [Ca2"] was calculated every 1.9 s from the
equation (Grynkiewicz et al., 1985):
[Ca2+], = (R - Rmi) X (S380,min/S380,,m,) X KD
(Rmax- R)
where KD is the affinity of fura-2 for Ca2" (224 nM at 37C)
and S380,min/S380,max is the ratio (P value) of the fluorescent
values obtained at 380 nm in the absence and presence of
saturating [Ca2+]j. The maximum and minimum R values
(Rmax and Rmin) were determined on separate coverslips under
saturating [Ca2+]i (achieved by increasing the extracellular
[Ca2+] to 20 mM followed by 10 gM ionomycin, pH 7.45) and
calcium-free (achieved using 8.3 mM EGTA immediately fol-
lowed by 25 jl of 1.0 M NaOH to compensate for the
decrease in pH, in the presence of 10pM ionomycin) condi-
tions respectively. Corrections for autofluorescence were
made by measuring the fluorescence produced by coverslips
that had not been loaded with fura-2. Where Ca2'-free con-
ditions were required experiments were performed in
nominally Ca2+-free buffer containing 0.1 mM EGTA.
Accumulation of [3H]-cyclic AMP
Adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumu-
lation was measured by prelabelling cell monolayers with
[3H]-adenine as described previously (Ruck et al., 1991).
Data analysis
Agonist and antagonist concentration-response curves were
fitted to a logistic equation using the non-linear regression
programme GraphPAD (ISI). Data are shown as means-
± s.e.mean. Statistical analysis was performed by use of
Student's unpaired t test. A P value < 0.05 was considered as
statistically significant. Rises in intracellular free [Ca2+] were
evaluated by importing the fluorescence data into the spread-
sheet AsEasyAs (TRIUS Inc). Basal [Ca2+]i levels were deter-
mined by calculating the mean of the ten data points
(measured every 1.9 s) before drug addition, whereas the
maximum Ca2+ signal was deemed to be the largest Ca2+
response obtained immediately after drug addition.
Apparent antagonist equilibrium dissociation constants
(KD) were estimated by a modification of the null method
described by Lazereno & Roberts (1987). Briefly, a
concentration-response curve to 2-chloroadenosine was
generated and a concentration (C; usually 10 1M) of 2-
chloroadenosine was chosen which gave a response greater
than 50% of the maximum agonist response. The concentra-
tion of antagonist (ICm) required to reduce the response to
this concentration (C) of 2-chloroadenosine by 50% was then
determined. The 2-chloroadenosine concentration-response
curve was fitted to a logistic equation as described above and
a concentration (C') identified which yielded a response
equivalent to 50% of that produced by concentration C (in
the absence of antagonist). The apparent KD was then deter-
mined from the relationship:
C/C' = IC50/KD + 1
Chemicals
Fura-2/AM, ionomycin and pertussis toxin were from Cal-
biochem. Histamine, mepyramine, forskolin, adenosine, 2-
chloroadenosine, 5'-N-ethylcarboxamidoadenosine (NECA),
N6-cyclopentyladenosine (CPA), indomethacin and 8-phenyl-
theophylline were purchased from Sigma and 8-cyclopentyl-
1,3-dipropylxanthine (DPCPX) from Research Biochemicals
Incorporated. AA861 (2,3,5-trimethyl-6-(12-hydroxy-5,10-
dodecadiynyl)1,4-benzoquinone) was a generous gift from
Takeda Chemicals Industries (Osaka, Japan). Dulbecco's
modified Eagle's medium and foetal calf serum (FCS) were
 Al-RECEPTORS AND [Ca2+]J IN DDT1MF-2 CELLS 87

from Northumbria Biologicals (U.K.). All other chemicals

were of analytical grade.

Results 2;-080 I

Adenosine receptor-stimulated increases in intracellular

Ca2+
Adenosine receptor-stimulation in the smooth muscle cell
line, DDT1MF-2, results in an increase in intracellular Ca2+
which appeared to be biphasic in nature. Adenosine (100 pM,
see Figure la) increased basal [Ca21], from 106 ± 11 nM to
217 ± 40 nM (n = 9, P< 0.05), within 20 s of application. The
response was fairly well maintained and declined slowly
towards basal levels in the presence of extracellular Ca2" (see
Figure la). Similar responses were obtained with 2-chloro-
adenosine (10 ftM, 86 ± 8 nM to 250 ± 25 nM, n = 6,
P< 0.05), N6-cyclopentyladenosine (1 MM, 98 ± 10 nM to
201 ± 18 nM, n = 4, P<0.05) and 5'-N-ethylcarboxamido-
adenosine (10IM, 100± 11 nM to 226±41 nM, n=3,
P< 0.05). Representative [Ca2]i profiles are shown in Figure
1. Concentration-response curves for CPA (EC50
= 19+ 7 nM, n = 5) and NECA (ECm = 770 ± 165 nM,
n = 4) are shown in Figure 2. Concentration-response curves
for adenosine and 2-chloroadenosine-stimulated calcium re-
sponses yielded EC50 values of 1.6 ± 0.6 MM (n = 5) and
1.3 ± 0.4 MM (n = 4), respectively. The rank order of agonist
potencies i.e. CPA> NECA> 2-chloroadenosine> adenosine
was indicative of the adenosine-receptor mediating increases
in [Ca2+]i being the A1 subtype. These values are in close
agreement with agonist potencies reported for the stimulation
of [3H]-inositol phosphate accumulation in DDT2MF-2 cells
(White et al., 1992). The class of adenosine-receptor involved
was further examined by using the selective Al-receptor
antagonist DPCPX (Bruns et al., 1987 a) and partially selec-
tive Al-receptor antagonist 8-phenyltheophylline. Inhibition
concentration-response curves were obtained under anta-
gonist equilibrium conditions (15 min preincubation) in the
presence of a constant 2-chloroadenosine concentration
(10 MM). The inhibition curves for DPCPX (ICm0 = 1.3 ±
0.2 nM, n = 3) and 8-phenyltheophylline (IC50 = 1.03 ±
0.3 MM, n = 4) are shown in Figure 3. This particular app-
roach enabled apparent equilibrium dissociation constants
(KD) to be calculated assuming a competitive interaction
between agonist and antagonist (see Methods). The KD
E
E*E40/
20-
0
-10 -9 -7 -8 -6 -5 -4
log [Agonist] (M)
Figure 2 Concentration-response curves for adenosine-receptor
agonists on increases in intracellular Ca2l concentration in
DDT1MF-2 cells. Responses to N6-cyclopentyladenosine (CPA: 0)
and N-ethylcarboxamidoadenosine (NECA: 0) are expressed as a
percentage of the maximal stimulation (expressed as an increase in
F340,/F380 ratio minus the basal fluorescence ratio). Curves were fitted
by use of a logistic equation as described under Methods. Data are
means and vertical lines show s.e.mean of five (CPA) or four
(NECA) experiments.
values obtained were 0.14 ± 0.02 nM (n = 3) and 112 ± 33 nM
(n =4) for DPCPX and 8-phenyltheophylline, respectively.
These values are again indicative of the involvement of the
Al-receptor subtype and are in agreement with antagonist KD
values for the inhibition of [3H]-cyclohexyladenosine binding
to the adenosine Al-receptor in rat brain membrane (Bruns et
al., 1987b).
The role of arachidonic acid metabolites
To assess the involvement of arachidonic acid metabolites i.e.
prostaglandins and leukotrienes in the adenosine-receptor
mediated increase in [Ca2+], we performed experiments using
the cyclo-oxygenase inhibitor indomethacin and the 5-
lipoxygenase inhibitor AA861 (Yoshimoto et al., 1982). The
rise in (Ca2+]i elicited by CPA (300 nM), in nominally Ca2"-
free buffer containing 0.1 mM EGTA, was slightly attenuated

a
250-1 250 b1 250fl
c

200- Aden 200 -

I Al

2 150- 150-
E
i 100- 100- 100-

50- 50- 50-

0 0- r -, 0 r
0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200
Time (s) Time (s) Time (s) Time (s)
Figure 1 Effect of adenosine-receptor stimulation on intracellular Ca2l concentration in fura-2 loaded DDT1MF-2 cells. Cells
were stimulated with the agonists (a) adenosine (Aden; 100 gM), (b) 2-chloroadenosine (2-CA; 10 gM), (c) N6-cyclopentyladenosine
(CPA; 1 gM) or (d) 5'-N-ethylcarboxamidoadenosine (NECA; 10 gM) in the presence of extracellular Ca2l (2 mM), added where
indicated.
88 J.M. DICKENSON & S.J. HILL

consequence of adenosine-receptor agonists augmenting the

inositol phospholipid response elicited by endogenous his-
tamine or noradrenaline (Schachter et al, 1992) leading to a
release of intracellular Ca2", we performed experiments in
the presence of the histamine HI-receptor antagonist
0
C80- mepyramine and the &x-adrenoceptor antagonist phen-
tolamine. Pretreatment of cells with mepyramine (100nM,
.Z60- Figure 4d) or phentolamine (300 nM, Figure 4e) did not
affect the response elicited by CPA (300 nM) in nominally
3 40-
Ca2"-free buffer containing 0.1 mM EGTA. In the presence
of mepyramine and phentolamine the CPA response was
107 ± 7.2% (n = 4) and 116 + 11% (n = 3) respectively of
20- that obtained in the absence of antagonist.

0 Role of extracellular Ct+ ions

-11 -10 -9 -8 -7 -6 -5
log [Antagonist] (M)
Figure 3 Concentration-response curves for adenosine-receptor
antagonists on increases in intracellular Ca2` concentration in
DDTIMF-2 cells. Antagonism produced by 8-cyclopentyl-1,3-
dipropylxanthine (DPCPX, 0) or 8-phenyltheophylline (8-PT: 0) of
the response to 10 M 2-chloroadenosine. The data are expressed as a
percentage of the response to 10AM 2-chloroadenosine (expressed as
an increase in F340/F380 ratio minus the basal fluorescence ratio).
Curves were fitted by use of a logistic equation as described under
Methods. Data are means and vertical lines show s.e.mean of three
(DPCPX) or four (8-PT) experiments.
by pretreatment of cells with AA861 (86 ± 2.1% of the max-
imum CPA response, n = 5, P< 0.05, cf Figure 4a and b). In
contrast, indomethacin (2 gM) did not effect the CPA-
induced rise in [Ca2]i (124 + 18% of the maximum CPA
response, n =4, Figure 4c). These data suggest that
adenosine-receptor stimulation, in nominally Ca2"-free buffer
containing 0.1 mM EGTA, can produce a direct release of
Ca2+ from intracellular stores which is independent of
arachidonic acid metabolites.
To eliminate the possibility that the rise in [Ca21]J is a
-4
The role of extracellular calcium in the overall response to
adenosine-receptor stimulation was examined by performing
experiments in nominally Ca2"-free buffer containing 0.1 mM
EGTA. Figure 5a shows a profile obtained by stimulating
adenosine-receptors with 300 nM CPA in the absence of ext-
racellular Ca2+. Clearly, the removal of extracellular Ca2`
results in a more transient response to CPA with [Ca2+]i
returning to basal levels approximately 150 s after stimula-
tion (also see Figures 5b and 7c). The attenuation of the
maintained phase of the response is clearly demonstrated if
the percentage of the maximum response (obtained using
300 nM CPA) remaining after 100 s is compared for
experiments performed in Ca2"-free and Ca2"-containing
buffer. In nominally Ca2"-free buffer containing 0.1 mM
EGTA the percentage of the maximum CPA response
remaining at 100 s is significantly lower (19.6 ± 4.7%, n = 6,
P<0.05) than that obtained in the presence of extracellular
Ca2+ (56.3 ± 3.2%, n = 6). Furthermore, if Ca2+ is re-
applied, after the cells have been stimulated with 300 nM
CPA in the absence of extracellular Ca2+, there is a rapid rise
in [Ca2+]i indicative of Ca2+ influx (see Figures 5b and 7c).
To dismiss the possibility that this rise in [Ca2J]i is simply a
consequence of fura-2 leakage into the extracellular medium
an experiment was performed (data not shown) in which

a b c d 0
4001 400 ,

350- 350 -

300- 300 -
CPA
250- I 250 - CPA
C
E 200- 200 -
:13
._

u150- 150 -

100- 100 -

50- 50 -

0- I -I 0-
100 200 0 100 200 0 100 200
Time (s)
Figure 4 Effects of arachidonic acid metabolism inhibitors and antagonists of the histamine H,-receptor and £-adrenoceptor on
adenosine-receptor stimulated increases in intracellular Ca2l concentration in DDTIMF-2 cells. Cells were stimulated in nominally
Ca2'-free buffer containing 0.1 mM EGTA with N6-cyclopentyladenosine (CPA) in the absence (a) or presence of 20 gM AA861 (b),
2 gM indomethacin (c), 100 nM mepyramine (d) or 300 nM phentolamine (e). Cells were incubated with the relevant inhibitor or
antagonist for 15 min before the addition of CPA. N6-cyclopentyladenosine (CPA: 300 nM) was added where indicated. Similar
results were obtained in at least three other experiments.

a
500- 2 mM CaCI2
_ 400- I I
C
c
E 300-
13 200-
100-
0i 6 1i0 260 300 460 50o
b
400 DPCPX
-
300-
E 200-
.5
MYho VI I o-
o 100-
0-
6 1bo 200 300 400 500
Time (s)
Figure 5 Effects of re-applying extracellular Ca2l (2 mM) in the
absence (a) and presence (b) of the Al-receptor antagonist (8-
cyclopentyl-1,3-dipropylxanthine (DPCPX), during experiments per-
formed in nominally Ca2l-free buffer and 0.1 mM EGTA.
N6-cyclopentyladenosine (CPA; 300 nM), DPCPX (100 nM) or
CaCl2 (2 mM) were added where indicated. Similar results were
obtained in at least five other experiments.
CPA was replaced with vehicle. There was no observable
increase in [Ca2+]i during this experiment, indicating that the
rise in [Ca2+], (after reapplying 2 mM CaCl2) shown in Figure
5a is a result of calcium entry into the cell. However, as
shown in Figure 5b, removing CPA from the receptor (with
100 nM DPCPX; applied 6 min before 2 mM CaCl2) atten-
uates the rise in [Ca2+]j observed when Ca2+ (2 mM) is re-
applied after the cells have been stimulated with CPA
(300 nM) in the absence of extracellular Ca2+. These data
suggest that the Ca' entry (influx) that occurs during the
later phase of the response to CPA is dependent on the
continued presence of CPA at the Al-receptor.
Pertussis toxin sensitivity of the Ca2+ response
We performed experiments with pertussis toxin (PTX) to
determine whether the two components of the adenosine
Al-receptor-mediated increase in [Ca2+],, i.e. intracellular
Ca2+ release and influx, are equally sensitive to inhibition by
PTX. Previous studies with DDTIMF-2 cells have demon-
strated that adenosine receptor agonists are able to inhibit
the formation of cyclic AMP induced by isoprenaline and
that PTX pretreatment reversed these effects (Gerwins et al.,
1990). In our DDTIMF-2 cells, the addition of CPA
inhibited the accumulation of [3HJ-cyclic AMP induced by
forskolin. CPA (300 nM) inhibited 93 + 4% (n = 3) of the
cyclic AMP accumulation induced by 10 M forskolin, with
an IC50 of 2.8 ± 0.5 nM (n = 3, see Figure 6). However,
pretreatment of cells with PTX (200 ng ml-' for 4 h)
attenuated the ability of CPA to inhibit forskolin-induced
[3H]-cyclic AMP accumulation (Figure 6). PTX pretreatment
(200 ng ml-' for 4 h) completely abolished the CPA-induced
release of intracellular Ca2+ and subsequent Ca2" influx
observed when Ca2+ is re-applied after the cells had been
stimulated with CPA in the absence of extracellular Ca2+
(compare c and d in Figure 7). In contrast, PTX pretreat-
ment did not attenuate histamine H,-receptor stimulated
intracellular Ca2+ release or Ca2+ influx (compare a and b in
Figure 7). The maximum peak response to histamine
(100 gM) obtained in Ca2+-free buffer and upon re-addition
Al-RECEPTORS AND [Ca2+]i IN DDTIMF-2 CELLS 89
600
log [CPA] (M)
6 Figure 6 Effects of N6-cyclopentyladenosine (CPA) and pertussis
toxin (PTX) on forskolin-induced cyclic AMP accumulation in
DDT1MF-2 cells. Control cells (0) and cells treated with PTX (0)
were incubated with 1Opgm forskolin for 10min in the presence of
6,00 different concentrations of CPA. Data are expressed as a percentage
of the response to 10gvm forskolin in the presence of PTX or CPA,
which was taken as 100%. Data are means and vertical lines show
s.e.mean of quadruplicate determinations in a single experiment.
Curves were fitted by use of a logistic equation as described under
Methods. Similar data were obtained in two other experiments.
of extracellular Ca"z (2 mM) in PTX treated cells were
133 ± 2% (n = 3) and 96 ± 8% (n = 3) respectively, of those
obtained with untreated cells. Furthermore, PTX pretreat-
ment had no effect on bradykinin (100 nM) induced release of
intracellular Ca2+ (95 ± 3% of the maximum response
measured in nominally Ca2'-free buffer containing 0.1 mM
EGTA; n = 3) or noradrenaline (100 gM) stimulated increases
in [Ca2+], measured in the presence of extracellular Ca2+
(107 ± 5% of the maximum response; n = 3).
Discussion
Recent studies have shown that adenosine A,-receptor activa-
tion in DDTMF-2 cells stimulates inositol phospholipid hyd-
rolysis and calcium mobilization from intracellular stores
(Gerwins & Fredholm, 1991; White et al., 1992). However,
other studies with DDTIMF-2 cells (Hoiting et al., 1990;
Schachter & Wolfe, 1992; Schachter et al., 1992) have sug-
gested that adenosine Al-receptor activation does not
stimulate the hydrolysis of inositol phospholipids or mobilize
intracellular calcium. Schachter et al. (1992) found that
adenosine Al-receptor activation potentiated the inositol
phospholipid response elicited by noradrenaline. However, in
the absence of noradrenaline adenosine Al-receptor activa-
tion caused only a minor stimulation of phospholipase C,
which was abolished in the absence of extracellular calcium.
The reasons for these conflicting results remain to be estab-
lished.
The data presented in this study clearly show that
adenosine-receptor activation in monolayers of the hamster
vas deferens derived smooth muscle cell line DDTIMF-2
stimulates a rapid and fairly well maintained increase in
[Ca2+]J. In the absence of extracellular Ca2+ ions
(experiments performed in nominally Ca2'-free buffer con-
taining 0.1 mM EGTA) the rise in [Ca2+], was transient in
nature (see Figure 4a) and is probably due to the release of
Ca2+ from intracellular stores. Furthermore, in our previous
study (White et al., 1992) we found that removal of extracel-
lular Ca2+ had no significant effect on the initial Ca2+ peak
elicited by 2-chloroadenosine (1O SM) compared with the re-
sponse obtained in Ca2"-containing buffer. This is in contrast
to histamine-induced changes in [Ca2J]i where the response in
90 J.M. DICKENSON & S.J. HILL

DDT1MF-2 cells is sensitive to inhibition by the 5-

500-
CaC12 2 mM lipoxygenase inhibitor AA861 (White et al., 1992). Thus, the
I1I calcium response may be secondary to the formation of
400- leukotrienes. Alternatively, CPA may augment responses to
300
endogenous noradrenaline or histamine (Schachter et al.,
E 1992). The data presented in Figure 4 clearly showed that in
*, 200- the absence of extracellular Ca2" ions (Ca2"-free buffer con-
(U taining 0.1 mM EGTA) and in the presence of the 5-
100- lipoxygenase inhibitor AA861 or the cyclo-oxygenase
inhibitor indomethacin, CPA still elicited significant increases
0- in [Ca2+]j. These data suggest that the adenosine-receptor
0 50 100 150 200 250

mediated release of intracellular Ca2+ is not dependent upon

tefn
b the products of arachidonic acid metabolism. In addition, the
Ouu
H CaC12 2 mm inclusion of mepyramine or phentolamine to block histamine
I
-
400- H,-receptors and x-adrenoceptors respectively, did not
attenuate the response to CPA, obtained in the absence of
E 300- extracellular Ca2+ ions. These data eliminated the possibility
E

.5 200-
that the rise in [Ca2+], is mediated via augmentation of
histamine or noradrenaline mediated calcium responses.
0
100- The adenosine-receptor mediated increase in [Ca21]J comp-
rises two distinct components: (1) release of Ca2+ from intra-
0o cellular storage sites (mobilization), which is probably secon-
50 100 150 200 250
dary to the production and action of inositol 1,4,5-
c trisphosphate (Berridge & Irvine, 1989), and (2) influx of
300- extracellular Ca2+ through Ca2" entry pathways in the
plasma membrane. Furthermore, the influx of extracellular
C 200-
Ca2+ into the cytoplasm is dependent upon continued
adenosine-receptor occupancy (receptor-mediated) since it is
E blocked by the Al-receptor antagonist DPCPX (see Figure
._5
cr 100-
5b). These data are similar to those obtained for the his-
tamine HI-receptor mediated increases in [Ca2J]i in
DDTMF-2 cells (Dickenson & Hill, 1992), although the
0-
relationship between adenosine-receptor activated Ca2" influx
0 50 100 150 200 250 and the refilling of intracellular Ca2" stores remains to be
established.
d The rank order of agonist potencies (CPA>NECA>2-
300- chloroadenosine> adenosine) is characteristic of the Al-
CPA CaCI2 2 mM
receptor. The involvement of the A, subtype was confirmed
by the sensitivity of the adenosine-receptor mediated calcium
c 200- response to inhibition by the A,-selective antagonist DPCPX.
E The rise in [Ca2+], is inhibited by concentrations of DPCPX
that are indicative of the adenosine Al-receptor, i.e. KD
0'r, 100- values in the order of 0.5 nM (Bruns et al., 1987a).
CJ There have been several reports in the literature of adeno-
sine-receptor activation stimulating increases in [Ca2+]j. Tran-
0 50 100 150 200 250 sient and pertussis toxin (PTX) sensitive Ca2` responses were
Time (s) demonstrated in rat mast cell line, RBL-2H3 and kidney
Figure 7 Effect of pertussis toxin (PTX) on histamine HI-receptor epithelial cells (Arend et al., 1988; 1989; Weinberg et al.,
and adenosine-receptor stimulated increases in intracellular Ca2l 1989; Ali et al., 1990). The rank order of agonist potencies
concentration in DDTIMF-2 cells. In all four experiments (a)-(d) suggested that in RBL-2H3 cells the Ca2+ response was
the cells were initially exposed to the appropriate agonist in mediated via A2-receptors. In contrast, the Ca2+ response
nominally Ca2"-free buffer containing 0.1 mM EGTA after which obtained with cultured rat mesangial cells (Olivera et al.,
exogenous Ca2l (2 mM) was re-applied. In experiments (b) and (d) 1992) was biphasic in appearance and comprised of an initial
cells were treated with PTX (200 ng ml-') for 4 h before measure- peak followed by a secondary increase which was smaller
ment of [Ca2+]i. The control experiments for histamine (a) and than the initial rise. In addition, the intracellular Ca2+ release
adenosine-receptor (c) stimulation were obtained on the same experi- was dependent upon extracellular Ca2+ since it did not occur
mental day.
Histamine (H; 100 tLM), N6-cyclopentyladenosine (CPA; 100 nM) or in Ca2'-free buffer. These data suggest that in other cell
CaCl2 (2 mM) were added where indicated. Similar results were preparations the adenosine-receptor stimulated rise in [Ca2+]i
obtained in two other experiments. occurs either via A2-receptors or requires the presence of
extracellular Ca2+. However, in DDTIMF-2 cells the res-
ponse is mediated via A,-receptors and can occur in the
absence of extracellular Ca2+ suggesting a coupling of the
Ca2+-free buffer is significantly lower than that obtained in adenosine-receptor to phospholipase C.
Ca2+-containing buffer (Dickenson & Hill, 1991; 1992). The finding that pertussis toxin pretreatment inhibits the
We have shown conclusively that adenosine-receptor adenosine Al-receptor induced rise in cytosolic Ca2+ (this
agonists can elicit increases in [Ca2+1] in the absence of study) and inositol phospholipid hydrolysis (White, T.E. &
extracellular Ca2+ (see Figures 4 and 5). However, there is Hill, S.J; unpublished observations) is consistent with the
some uncertainty as to whether the hydrolysis of inositol involvement of a PTX sensitive G-protein in adenosine Al-
phospholipids (White et al., 1992) and the mobilization of receptor-mediated events (Stiles, 1992). These data contrast
intracellular Ca2+ are a consequence of adenosine-receptors with those obtained in guinea-pig uterine smooth muscle
being directly coupled to phospholipase C via a G-protein or where adenosine Al-receptor stimulation of inositol phos-
whether they are mediated via indirect mechanisms. For pholipid hydrolysis is not blocked by PTX pretreatment
example, the inositol phospholipid response to CPA in (Schiemann et al., 1991). In addition, the inositol phosphate

response in guinea-pig uterine smooth muscle is cyclo-
oxygenase sensitive whereas, in DDT1MF-2 cells
indomethacin was without significant effect (White et al.,
1992).
Recent studies using cloned human 5-HTIA receptors ex-
pressed at high concentrations in transfected HeLa cells have
revealed that they are able to couple both negatively to
adenylate cyclase (which is the normal effector system for
5-HTIA receptors) and positively to phospholipase C (Bod-
deke et al., 1992). In view of the high level of adenosine
Al-receptors expressed in DDT1MF-2 cells (Gerwins et al.,
1990; Ramkumar et al., 1990) it may be that in this partic-
ular cell line adenosine Al-receptors can couple, via different
G-proteins, both negatively to adenylate cyclase (as indicated
by the ability of CPA to inhibit forskolin-induced [3H]-cyclic
AMP accumulation) and positively to phospholipase C (as
indicated by CPA mediated inositol phospholipid hydrolysis
and calcium mobilization). Further evidence for this proposal
is provided by comparing the EC50 values obtained for CPA-
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(Received July 8, 1992
Revised August 20, 1992
Accepted August 28, 1992)