A1 adenosine
receptor-mediated
Ins(1,4,5)P3
generation in allergic rabbit airway smooth muscle
Worku
Abebe and
S. Jamal
Mustafa
Department of Pharmacology, School of Medicine, East Carolina
University, Greenville, North Carolina 27858
 |
ABSTRACT |
The signal transduction pathway for
A1 adenosine receptor in airway
smooth muscle from allergic rabbits was studied by investigating the
effect of the selective A1
adenosine-receptor agonist
N6-cyclopentyladenosine
(CPA) on tissue levels of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]
measured by protein binding assay. CPA caused a rapid, transient, and
concentration-dependent elevation of
Ins(1,4,5)P3 in
airways from allergic rabbits. The agonist also produced a concentration-dependent contraction of the airway preparations from
these animals. Both the
Ins(1,4,5)P3 and
contractile responses generated by CPA were attenuated by the
phospholipase C (PLC) inhibitor U-73122, indicating the coupling of
these responses to PLC. The CPA-induced
Ins(1,4,5)P3
production observed in the allergic rabbit tissues was also inhibited
by the adenosine-receptor antagonist
8-( p-sulfophenyl)-theophylline,
suggesting that the effect was mediated by
A1 adenosine receptors. On the
other hand, the A2
adenosine-receptor agonist CGS-21680 was ineffective in altering the
tissue concentration of
Ins(1,4,5)P3,
indicating that A2 adenosine
receptors may not be involved in the activation of PLC in the allergic
rabbit airway smooth muscle. In this preparation, the
Gi-Go
inhibitor pertussis toxin (PTX) attenuated the CPA-induced Ins(1,4,5)P3
accumulation, providing evidence that the generation of
Ins(1,4,5)P3 by
A1 adenosine-receptor stimulation
is coupled to a PTX-sensitive G protein(s). The results suggest that
activation of A1 adenosine
receptors in allergic rabbit airway smooth muscle causes the production
of Ins(1,4,5)P3
via a PTX-sensitive G protein-coupled PLC, and this signaling mechanism
may be involved, at least in part, in the generation of contractile
responses. It is hypothesized that this process may contribute to
adenosine-induced bronchoconstriction in allergic asthma.
asthma; N6-cyclopentyladenosine; phospholipase C; G protein; airway responsiveness; inositol
1,4,5-trisphosphate
 |
INTRODUCTION |
ASTHMA IS A DISEASE characterized by a variety of
respiratory conditions, including airway inflammation,
hyperresponsiveness, and obstruction. It affects ~10% of the
population in industrialized countries, and this figure is anticipated
to increase in the next decade (12, 26). Although the cause of asthma
remains obscure, a number of stimuli have been recognized to trigger
the airway hyperresponsiveness associated with the disease. This has
been supported by several lines of evidence from clinical and
experimental observations. In humans, adenosine has been shown to
induce bronchoconstriction in asthmatic but not in normal subjects
(16). Airway smooth muscle isolated from individuals with allergic
asthma have also been demonstrated to be hyperreactive to adenosine
(9). In addition, the concentration of adenosine in bronchoalveolar
lavage fluid of asthmatic subjects has been observed to be elevated
(17). In a rabbit model of asthma that simulates the human condition of
the disease, adenosine has been shown to cause bronchoconstriction and
bronchial hyperresponsiveness (4, 6, 7). Similarly, the nucleoside has
been observed to induce contraction of airway smooth muscle isolated
from this allergic rabbit model (4, 6). The bronchial lavage
concentration of adenosine has also been reported to be elevated in
this model (5). On the other hand, all these characteristics associated
with the disease have been found to be lacking in age-matched
nonallergic rabbits (4-7).
A further study (6) conducted in our laboratory demonstrated that the
adenosine-induced bronchoconstriction observed in the allergic rabbit
model of asthma was mediated via
A1 adenosine receptors. El-Hashim
et al. (18) from another laboratory also reported similar
results with this model. These in vivo results were also confirmed by
in vitro experiments with the use of isolated airway smooth muscle
preparations from allergic rabbits (4, 6). In addition, specific
A1 adenosine-receptor binding
sites have been shown in the lung plasma membranes from allergic
rabbits (6). Furthermore, A1
adenosine receptor-mediated contractile responses observed in allergic
rabbit airway smooth muscle was found to be linked to increased
intracellular levels of calcium (4).
It is generally agreed that calcium-mobilizing agonists elicit
contraction of airway smooth muscle by mechanisms that involve activation of phospholipase C (PLC) via receptor-coupled GTP-binding proteins (G proteins) (13). PLC consists of a family of related isozymes with distinct tissue distribution and biochemical requirements (36, 37). Activation of this enzyme induces the breakdown of plasma
membrane phosphatidylinositol 4,5-bisphosphate, resulting in the
generation of the second messengers inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]
and 1,2-diacylglycerol (DAG) (36).
Ins(1,4,5)P3 can
mobilize calcium from nonmitochondrial intracellular stores, such as
sarcoplasmic reticulum, and this effect has been suggested to
contribute to the initiation of contraction. DAG, on the other hand,
activates protein kinase C, which is believed to be involved in the
maintenance of contractile responses together with calcium entering the
cells from extracellular sources. The present study was undertaken to
further understand the mechanism of action of adenosine in asthma by
investigating the effect of A1
adenosine-receptor stimulation on PLC-induced generation of
Ins(1,4,5)P3 in
airway smooth muscle from the allergic rabbit model. The
N6-substituted
adenosine analog
N6-cyclopentyladenosine
(CPA) was used as an A1
adenosine-receptor agonist. This compound was previously reported to be
a potent bronchoconstrictor in this model of asthma and is considered
to be a useful marker of airway hyperresponsiveness (4, 6, 7).
 |
MATERIALS AND METHODS |
Induction of asthma. Asthma was
induced in allergic rabbits according to the established methods used
in our laboratory (7). Briefly, New Zealand White rabbit littermates
free of Pasteurella were immunized
intraperitoneally within 24 h of birth with 312 allergen
units of dust mite allergen extract suspended in 10% kaolin once every week for the first month and then biweekly for the
following 3 months. Such immunized rabbits preferentially generate
allergen-specific IgE antibody and positively respond to aeroallergen
challenges with early- and late-phase asthmatic responses as well as
hyperresponsiveness (7, 25). This model closely simulates events
occurring in human asthmatics (25). Age-matched nonimmunized
littermates treated under identical conditions were used as controls,
hereafter referred to as nonallergic rabbits. To evaluate airway
sensitivity to adenosine, increasing doses of the nucleoside ranging
from 0.16 to 20 mg/ml in 0.9% saline were aerosolized in anesthetized
rabbits as reported previously (6, 7). Total lung resistance and
dynamic compliance (Cdyn) were
recorded on an automated pulmonary mechanics respiratory analyzer
(model 6, Buxco Electronics, Troy, NY). From this, the concentration of
adenosine producing a 50% reduction in
Cdyn from baseline
(PC50) was determined for each
rabbit (6, 7).
Ins(1,4,5)P3 assay.
Airways were removed from allergic and nonallergic rabbits and cleaned
of fat and parenchymal tissue down to the tertiary branches. Primary,
secondary, and tertiary rings were prepared and denuded of the
epithelium as previously described (4, 6). The rings were mounted in
isolated tissue baths containing gassed (95%
O2-5%
CO2) Krebs-Henseleit solution
with a composition of (in mM) 118 NaCl, 4.8 KCl, 1.2 MgSO4, 1.2 KH2PO4,
25 NaHCO3, 11 glucose, and 2.5 CaCl2 (pH 7.4, 37°C). After a
1-h equilibration period, the tissues were treated with 30 mM KCl for 5 min. Tissues were then washed and further equilibrated for 40 min.
During the equilibration periods, the bathing solution was changed
every 15-20 min. The airway rings were then incubated with the
A1 adenosine-receptor agonist CPA
for various times (10-30 s) and at different concentrations (10
7 to
10
4 M) in the absence or
presence of PLC inhibitors
(10
5 M) neomycin
and
1-[6-((17
-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione (U-73122; 20-min preincubation), an adenosine-receptor antagonist [10
5 M)
8-( p-sulfophenyl)-theophylline
(8-SPT); 20-min preincubation], and a
Gi-Go
inhibitor [100 ng/ml of pertussis toxin (PTX); 3-h preincubation] (31). For comparison purposes, a few key
experiments were also performed in tertiary airway rings with the use
of the A2 adenosine-receptor
agonist
2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680). After incubation with drugs, the rings were frozen in
liquid nitrogen and stored at
80°C until assayed for
Ins(1,4,5)P3 levels as described by Abebe and MacLeod (1, 2). Control tissues that
were not treated with drugs were also frozen to measure basal
Ins(1,4,5)P3
contents. The frozen rings were weighed, pulverized, and homogenized in
6% TCA, followed by centrifugation. The supernatants were treated four
times with four volumes of ether to remove the TCA.
Ins(1,4,5)P3
levels in the aqueous extracts were determined with protein-binding
assay kits (1, 2).
Contractility studies. Primary,
secondary, and tertiary airway tissues isolated from allergic rabbits
and prepared as epithelium-denuded rings as described in
Ins(1,4,5)P3
assay were mounted in isolated tissue baths containing
Krebs-Henseleit solution (95% O2-5%
CO2, pH 7.4, 37°C) for
measurement of isometric tension (3, 6). The rings were placed under a
resting tension of 2 g for primary rings and 1 g for secondary and
tertiary rings. After equilibrating for 1 h, the tissues were
challenged with KCl (30 mM) until reproducible contractile responses
were obtained. After an additional equilibration period of 40 min,
cumulative concentration-response curves to CPA
(10
8 to
10
4 M) were obtained in all
the primary, secondary, and tertiary airway rings. For determination of
the involvement of PLC in CPA-induced contractile responses, the
concentration-response curves for the agonist were repeated in the
presence of neomycin (10
5
M) or U-73122 (10
5 M).
Tissues were washed for 1 h between CPA concentration-response curves
and incubated in the presence of the inhibitors for 20 min before CPA
was added. In preliminary experiments, it was confirmed that no changes
in the contractile responses of the airways to CPA occurred with time.
At the end of each experiment, tissues were blotted dry and weighed.
Materials. House dust mite extract was
purchased from Berkeley Biologicals (Berkeley, CA).
Ins(1,4,5)P3 kits
were procured from Amersham Radiochemical (St. Louis, MO). CPA,
CGS-21680, and 8-SPT were purchased from RBI (Natick, MA). Neomycin was
purchased from Sigma (St. Louis, MO). PTX was obtained from
List Biological Laboratories (Campbell, CA). U-73122,
1-[6-((17
-3-methoxyestra-1,3,5-(10)-trien-17-yl)amino)-hexyl]-2,5-pyrrolidinedione (U-73343), and tricyclodecane-9-yl-xanthogenate (D-609) were
from Calbiochem (La Jolla, CA).
Data analysis. The levels of
Ins(1,4,5)P3 were
calculated as picomoles per milligram of tissue weight. Contractile
responses to CPA are expressed as the increase in tension (in g) in
response to each concentration of the agonist per milligram of tissue
weight. pD2 (
log
EC50) values, which were used to
assess the sensitivities of the airway smooth muscle to CPA, were
determined from the concentration-response curves (3). Results are
reported as geometric means ± SE calculated with a STAT-MATE
program. Data were compared with the Student's t-test and considered to be
significant if P < 0.05.
 |
RESULTS |
As reported previously (6, 7), at the time of death, the majority of
dust mite-sensitized rabbits revealed bronchial hyperresponsiveness to
adenosine. The PC50 for adenosine
in these animals was 2.96 ± 0.45 mg/ml
(n = 15). Age-matched nonallergic rabbits demonstrated a much higher
PC50 at values > 20 mg/ml.
CPA-induced Ins(1,4,5)P3
generation.
The time course of
Ins(1,4,5)P3
accumulation in response to CPA in primary, secondary, and tertiary
tracheal rings from allergic and control rabbits is shown in Fig.
1. In all three preparations from both
groups of animals, CPA (10
5
M) caused a rapid rise in
Ins(1,4,5)P3
contents that reached a peak 10 s after administration of the agonist.
The levels of Ins(1,4,5)P3 then
declined with time, returning close to basal levels 20 s after CPA
addition, and continued to decline slightly over a period of 30 s.
However, in all airways from nonallergic rabbits, the peak
Ins(1,4,5)P3
levels in response to CPA were not significantly greater than the
corresponding basal levels. On the other hand, the CPA-induced maximum
generation of the polyphosphate in all the primary, secondary, and
tertiary rings from allergic rabbits was significantly higher than the
corresponding values measured in the absence of the agonist. Basal
Ins(1,4,5)P3
contents in the three types of airways from allergic rabbits were
similar to those assessed in corresponding tissues from nonallergic
rabbits. However, the maximum
Ins(1,4,5)P3
levels measured in the secondary and tertiary airway preparations from
allergic rabbits in response to CPA were significantly greater than the
peak Ins(1,4,5)P3
levels attained in response to the agonist in corresponding nonallergic tissues. The concentrations of
Ins(1,4,5)P3 did
not differ between nonallergic and allergic rabbit airways at any other
time points of incubation with CPA (Fig. 1).

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 1.
Time course of
N6-cyclopentyladenosine
(CPA; 10 5 M)-induced
inositol 1,4,5-trisphosphate
[Ins(1,4,5)P3]
generation in primary, secondary, and tertiary branches of airway
smooth muscle from control ( ) and allergic ( ) rabbits. Each point
is mean ± SE of 4-11 different observations. Significantly
different (P < 0.05) from:
* corresponding basal value;
# corresponding control peak
value.
|
|
Figure 2 illustrates the CPA
concentration-response curves for
Ins(1,4,5)P3
assessed in primary, secondary, and tertiary tracheal rings from
allergic rabbits at peak
Ins(1,4,5)P3
accumulation time (10 s). In all the three preparations, CPA induced
concentration-related (10
7
to 10
4 M) increases in
Ins(1,4,5)P3
production, with pD2 values of
5.20 ± 0.11, 5.08 ± 0.09, and 5.13± 0.06 for primary,
secondary, and tertiary rings, respectively.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 2.
Concentration-response curves for CPA-induced
Ins(1,4,5)P3
generation in airway smooth muscle from allergic rabbits.
[CPA], concentration of CPA. Tissues were incubated with
CPA for 10 s before reaction was terminated. b, Basal
Ins(1,4,5)P3
contents determined before CPA was added to tissues. Each point is mean ± SE of 4-8 different observations. * Significantly
different from corresponding basal value
(P < 0.05).
|
|
Preincubation of tertiary airway smooth muscle rings from allergic
rabbits with the adenosine-receptor antagonist 8-SPT
(10
5 M) significantly
diminished the accumulation of
Ins(1,4,5)P3 caused by CPA (10
5 M; Fig.
3). However, 8-SPT had no significant
effect on the basal concentration of
Ins(1,4,5)P3 in
this tissue.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of
8-(p-sulfophenyl)-theophylline (8-SPT;
10 5 M) on CPA
(10 5 M)-induced
Ins(1,4,5)P3
generation in tertiary branch of airway smooth muscle from allergic
rabbits. Tissues were pretreated with 8-SPT for 20 min before being
incubated with CPA for 10 s. Each bar is mean ± SE of 4 different
observations. * Significantly different from all other values
(P < 0.05).
|
|
In contrast to the responses of the airways to CPA, incubation of
tertiary airways with the A2
adenosine-receptor agonist CGS-21680
(10
5 M) for 5-20 s did
not produce a significant effect on the accumulation of
Ins(1,4,5)P3 in
either allergic or nonallergic rabbits (Fig. 4).

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 4.
Time course of CGS-21680
(10 5 M)-induced
Ins(1,4,5)P3
generation in tertiary branch of airway smooth muscle from control
( ) and allergic ( ) rabbits. Each point represents mean ± SE
of 4-6 different observations.
|
|
Pretreatment of primary, secondary, and tertiary airway preparations
from allergic rabbits with the phospholipase C inhibitor neomycin
(10
5 M) did not produce a
significant effect on CPA
(10
5 M)-induced
Ins(1,4,5)P3
generation (Fig. 5). Neomycin also did not
markedly alter the basal
Ins(1,4,5)P3
concentration of the tissue. On the other hand, U-73122
(10
5 M), a newly
synthesized PLC inhibitor, abolished the
Ins(1,4,5)P3 production elicited by CPA
(10
5 M) in all three types
of rings from allergic rabbits (Fig. 6). However, U-73122 also did not have a significant effect on basal levels
of
Ins(1,4,5)P3.

View larger version (41K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of neomycin (10 5 M)
on CPA (10 5 M)-induced
Ins(1,4,5)P3
generation in primary, secondary, and tertiary branches of airway
smooth muscle from allergic rabbits. Tissues were pretreated with
neomycin for 20 min before being incubated with CPA for 10 s. Each bar
is mean ± SE of 4-5 different observations.
* Significantly different from basal and neomycin alone-treated
values within the group (P < 0.05).
|
|

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of U-73122 (10 5 M)
on CPA (10 5 M)-induced
Ins(1,4,5)P3
generation in airway smooth muscle from allergic rabbits. Tissues were
pretreated with U-73122 for 20 min before being incubated with CPA for
10 s. Each bar is mean ± SE of 4-6 different observations.
* Significantly different from all other values within the group
(P < 0.05).
|
|
Pretreatment of primary, secondary, and tertiary airway rings from
allergic rabbits with the
Gi-Go
inhibitor PTX caused a significant attenuation of
Ins(1,4,5)P3
generation evoked by CPA (10
5 M; Fig.
7). PTX, however, did not produce an effect
on basal levels of
Ins(1,4,5)P3 in
any of the tissues.

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 7.
Effect of pertussis toxin (PTX; 100 ng/ml) on CPA
(10 5 M)-induced
Ins(1,4,5)P3
generation in airway smooth muscle from allergic rabbits. Tissues were
pretreated with PTX for 3 h before being incubated with CPA for 10 s.
Each bar is mean ± SE of 4-9 different observations.
* Significantly different from all other values within the group
(P < 0.05).
|
|
CPA-induced contractile responses. As
described previously (5, 6), the addition of CPA in a cumulative manner
(10
8 to
10
4 M) produced
concentration-dependent increases in the contractile responses of
primary, secondary, and tertiary rings isolated from allergic rabbits
(Figs. 8 and
9) . Incubation of the tissues with 10
5 M neomycin
produced no significant effects on their contractile responsiveness to
CPA as reflected by the maximum responses and pD2 values (Fig. 8, Table
1). However, as with the
Ins(1,4,5)P3 data
(Fig. 6), pretreatment of primary, secondary, and tertiary airways from
allergic rabbits with U-73122
(10
5 M) caused a
significant inhibition of the contractile effect of CPA, although the
pD2 values for the agonist were
not significantly altered by the inhibitor (Fig. 9, Table
2).

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 8.
Effect of neomycin (10 5 M)
on CPA-induced contraction of airway smooth muscle from allergic
rabbits. Tissues were incubated with ( ) or without ( ) neomycin
for 20 min before addition of CPA. Each point represents mean ± SE
of 4-8 different observations.
|
|

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 9.
Effect of U-73122 (10 5 M)
on CPA-induced contraction of airway smooth muscle from allergic
rabbits. Tissues were incubated with ( ) or without ( ) U-73122 for
20 min before addition of CPA. Each point represents mean ± SE of 4 different observations.
|
|
View this table:
[in this window]
[in a new window]
|
Table 1.
Effect of neomycin on CPA pD2 values and maximum
contractile responses in airway smooth muscle rings from allergic
rabbits
|
|
View this table:
[in this window]
[in a new window]
|
Table 2.
Effect of U-73122 on CPA pD2 values and maximum
contractile responses in airway smooth muscle rings from allergic
rabbits
|
|
 |
DISCUSSION |
In the present study, we have presented evidence that the
A1 adenosine-receptor agonist CPA
induces a rapid, transient, and concentration-dependent elevation in
Ins(1,4,5)P3
contents in allergic rabbit airway smooth muscle. However, the agonist
was unable to produce such an effect in airways from nonallergic
rabbits. These findings, therefore, suggest that activation of
A1 adenosine receptors results in
the production of
Ins(1,4,5)P3 in
allergic but not in normal rabbit airway smooth muscle. The involvement of adenosine receptors in this process is further supported by our
observation that the commonly used adenosine-receptor antagonist 8-SPT
(3) could selectively inhibit the CPA-induced
Ins(1,4,5)P3 production in this tissue. Because the
A2 adenosine-receptor agonist CGS-212680 did not elicit
Ins(1,4,5)P3
generation in either allergic or nonallergic rabbits,
A2 receptors may not be involved
in this mechanism. Consistent with the
Ins(1,4,5)P3
data, we also found that CPA could cause contraction of airway smooth
muscle from allergic but not from nonallergic rabbits. In both the
allergic and nonallergic tissues, CGS-21680 was also ineffective in
inducing contraction of all three types of airway tissues (data not
shown). Although the contractile data presented here are similar to
those previously reported (4, 6), the observation of enhanced production of
Ins(1,4,5)P3 in
the allergic rabbit airways in response to
A1 adenosine-receptor stimulation
is the first of its kind, although activation of this receptor in
DDT1 MF-2 smooth muscle cells by
other investigators (19) has also been shown to be associated with
increased generation of
Ins(1,4,5)P3. The
fact that the PLC inhibitor U-73122 could attenuate both the
CPA-induced contraction and
Ins(1,4,5)P3
generation in rabbit airways suggests that both of these responses are
mediated, at least in part, via PLC-linked mechanisms. Because the
contractile responses generated by CPA could not be inhibited by the
phosphatidylcholine-specific PLC inhibitor D-609 (33) (data not shown),
it is believed that the CPA-induced responses are mediated via
phosphatidylinositol-specific PLC. U-73122 is a novel aminosteroid that
has been reported by others to be a selective inhibitor of
phosphatidylinositol-specific PLC in platelets (10), neutrophils (35),
neuroblastoma (38), and guinea pig airway smooth muscle (32). The
current data obtained with allergic rabbit airways provide evidence for
the existence of an additional biological system containing at least
this PLC, which is susceptible to the inhibitory action of U-73122. On
the other hand, the lack of alteration of basal
Ins(1,4,5)P3
contents by U-73122 and the ineffectiveness of its nonactive analog
U-73343 (25) on contractile responses (data not shown) confirm the
absence of nonspecific action of the inhibitor on the allergic rabbit tissues. The failure of neomycin to attenuate the contractile as well
as the
Ins(1,4,5)P3
responses indirectly indicates that both of these events share a common
cellular mechanism, in this case, a mechanism that is not susceptible
to inhibition by this compound. Neomycin has also been reported to be
ineffective in attenuating certain agonist-induced contractions and
phosphatidylinositol hydrolysis (including those produced by
carbamylcholine and histamine) in guinea pig trachea (14), although
many studies (15, 28, 29, 31, 32, 34) have indicated otherwise in a
number of biological systems. The reason for these inconsistent
observations is unclear, but there is the suggestion that this could be
related to differences in the compartmentalization of receptor-coupled phosphatidylinositol 4,5-bisphosphate to a domain that is inaccessible to neomycin (14).
In the present investigation, the rapid increase in formation of
Ins(1,4,5)P3
observed in the allergic airway smooth muscle in response to CPA is in
close agreement with the hypothesis that Ins(1,4,5)P3 may
be responsible for the initiation of agonist-induced tension
development in this tissue (13). On the other hand, the fact that the
production of the second messenger caused by CPA was transient suggests
that it may not directly contribute to the maintenance of the
contractile response (4, 13, 24). In this respect, the influx of
extracellular calcium and protein kinase C activation by DAG, which is
also partly generated by hydrolysis of phosphatidylinositol
4,5-bisphosphate, may have a role in the maintenance of contraction (4,
13). It is believed that whereas the augmented formation of
Ins(1,4,5)P3 in
response to CPA was due to the activation of PLC, the decrease in the
levels of the polyphosphate after an initial increase could have
resulted from its metabolism to inositol 1,4-bisphosphate
and/or inositol 1,3,4,5-tetrakisphosphate by activated
Ins(1,4,5)P3
5-phosphatase and/or
Ins(1,4,5)P3
3-kinase present in the cytosol (8, 21). The decline in
Ins(1,4,5)P3
levels observed does not appear to be related to a desensitization
phenomenon because this was not recorded in the CPA
concentration-response curves for
Ins(1,4,5)P3 production.
Comparison of the concentration-response curves for CPA indicates that
the pD2 values for
Ins(1,4,5)P3
production in the three types of airway rings were less than the
corresponding pD2 values for
contraction. Similar discrepancies between
pD2 values for agonist-induced
contractile and biochemical responses have been reported by other
investigators for various smooth muscle preparations (22, 39). Several
possible explanations can be forwarded for this observation. First, the
assay used to measure
Ins(1,4,5)P3 may
not be as sensitive as that which detected the contractility changes.
Second, there could be amplification between second messenger production and contractile responses, such as agonist-induced enhancement of calcium sensitivity of contractile proteins (22, 39).
Last, there may be involvement of PLC-independent mechanisms and/or a greater efficiency of coupling between
A1 adenosine-receptor activation
and the influx of extracellular calcium in generating contractile
responses to submaximal concentrations of CPA (27).
In an attempt to assess the involvement of G proteins in
A1 adenosine receptor-mediated
Ins(1,4,5)P3
generation in the allergic rabbit airway smooth muscle, we determined
the effect of PTX on CPA-induced production of the second messenger.
The results reveal that PTX inhibited
Ins(1,4,5)P3
formation elicited by CPA in this smooth muscle. This observation
suggests that in the allergic rabbit smooth muscle CPA can evoke, at
least in part, the generation of
Ins(1,4,5)P3
through PTX-sensitive pathways. Because PTX is known to ADP-ribosylate
Gi and
Go proteins (20, 23), it is likely
that these proteins may be involved in the coupling of the
A1 receptor to PLC in this tissue
model. In support of this, it has been previously shown that increased
intracellular levels of
Ins(1,4,5)P3
caused by the activation of A1
adenosine receptors by CPA and adenosine in
DDT1 MF-2 smooth muscle cells are
mediated via PTX-sensitive G proteins (19).
The contractile data demonstrate that the CPA-induced responses tended
to be more pronounced from peripheral to central airways. This
observation confirms previous results from our laboratory (6), further
indicating that the smooth muscle surrounding smaller airways is more
sensitive to A1 adenosine-receptor
stimulation. However, unlike the contractile responses, the
corresponding changes in
Ins(1,4,5)P3
levels of the tissues were smaller, although a similar trend of
enhancement was detected. The reason for this discrepancy between the
biochemical and physiological responses is not clear. However, by
analogy with vascular preparations (11), one possible explanation could
be that as the size of the airways decreases, the contractile response
becomes more and more dependent on extracellular calcium rather than on
Ins(1,4,5)P3-releasable intracellular calcium.
In our study, the contribution of non-smooth muscle to the changes in
Ins(1,4,5)P3
contents was assumed to be minimal. This is because a previous study by
Langlands et al. (24) demonstrated that the levels of
Ins(1,4,5)P3
present in cartilage, which is a major component of the airway mass,
are below the level of detection for the assay system used here.
Furthermore, because the epithelium was removed from the tissues in the
present experiments, its contribution to
Ins(1,4,5)P3
contents was minimized. Therefore, the changes seen in
Ins(1,4,5)P3 were
the result of the action of CPA on the airway smooth muscle.
The present results could be relevant to the pathophysiology of asthma
in several aspects. Endogenous adenosine has been shown to be increased
in allergic asthma both in the rabbit model we used and in human
asthmatics (5, 17). Whereas a basal value of 2.87 µM was reported in
bronchoalveolar lavage fluid of allergic rabbits, the basal
concentration of the nucleoside in nonallergic animals was found to be
below detection level (5). In humans, the reported basal adenosine
contents were 2.55 and 0.72 µM in asthmatic and normal subjects,
respectively (17). The demonstration of an increased concentration of
adenosine in allergic asthma provides evidence for the role of
endogenous adenosine in provoking bronchoconstriction in asthma. This
observation is supported by the fact that the adenosine-receptor
antagonist theophylline is efficacious in the treatment of asthma, and
bronchospasm is one of the potential side effects of the
adenosine-uptake inhibitor dipyridamole when used in thallium
myocardial perfusion imaging (30). However, the reason(s) for the
increased adenosine concentration in allergic asthma and its source(s)
are unclear. In its action as a bronchoconstrictor, it is likely that
the nucleoside may, at least in part, act directly on the airway smooth
muscle adenosine receptors. In support of this, we have documented that
in airway preparations from allergic rabbits no inflammatory cells,
including mast cells, could be detected histologically and that the
mast cell secretagogue compound 48/80 was without effect on the
responses of the airways to adenosine-receptor agonists (S. Ali and S. J. Mustafa, unpublished data). On the basis of the CPA
data we obtained in the present study, it can therefore be speculated
that at least part of the endogenous adenosine-induced airway smooth
muscle reactivity under allergic conditions is linked to
phosphatidylinositol-specific PLC activity. This implies the
possibility that inhibitors of this enzyme can have the potential for
developing antiasthma drugs.
In conclusion, our results demonstrate that CPA via
A1 adenosine receptors induces
PLC-mediated
Ins(1,4,5)P3
generation and contraction in allergic rabbit airway smooth
muscle. This coupling process involves PTX-sensitive G proteins. The
results suggest that PLC activation may play an important role in
adenosine-induced bronchoconstriction in asthma and such an
understanding can be useful for possible therapeutic exploitation of
the signaling pathway.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Shahid Ali and Weixi Qin for expert technical
assistance. We also thank Pam Wynne for typing the manuscript.
 |
FOOTNOTES |
This work was supported by National Heart, Lung, and Blood Institute
(NHLBI) Grant HL-50049; the American Lung Association of North
Carolina; and a Minority Investigator Research Supplement Award from
the NHLBI to W. Abebe.
Present address of W. Abebe: Dept. of Oral Biology and Maxillofacial
Pathology, School of Dentistry, Medical College of Georgia, Augusta, GA
30912.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: S. J. Mustafa, Dept. of Pharmacology,
School of Medicine, East Carolina Univ., Greenville, NC 27858.
Received 9 February 1998; accepted in final form 24 July 1998.
 |
REFERENCES |
1.
Abebe, W.,
and
K. M. MacLeod.
Influence of diabetes on norepinephrine-induced inositol 1,4,5-trisphosphate levels in rat aorta.
Life Sci.
49:
PL85-PL90,
1991[Medline].
2.
Abebe, W.,
and
K. M. MacLeod.
Augmented inositol phosphate production in mesenteric arteries from diabetic rats.
Eur. J. Pharmacol.
225:
29-36,
1992[Medline].
3.
Abebe, W.,
S. Makujina,
and
S. J. Mustafa.
Adenosine receptor-mediated relaxation of porcine coronary artery in presence and absence of endothelium.
Am. J. Physiol.
266 (Heart Circ. Physiol. 35):
H2018-H2025,
1994[Abstract/Free Full Text].
4.
Ali, S.,
W. J. Metzger,
and
S. J. Mustafa.
Simultaneous measurement of cyclopentyladenosine-induced contraction and intracellular calcium in bronchial rings from allergic rabbits and its antagonism.
J. Pharmacol. Exp. Ther.
278:
639-644,
1996[Abstract].
5.
Ali, S.,
S. J. Mustafa,
A. G. Driver,
and
W. J. Metzger.
Release of adenosine in bronchoalveolar lavage fluid following allergen bronchial provocation in allergic rabbits (Abstract).
Am. Rev. Respir. Dis.
143:
A417,
1991.
6.
Ali, S.,
S. J. Mustafa,
and
W. J. Metzger.
Adenosine-induced bronchoconstriction and contraction of airway smooth muscle from allergic rabbits with late-phase airway obstruction: evidence for an inducible adenosine A1 receptor.
J. Pharmacol. Exp. Ther.
268:
1328-1334,
1994[Abstract].
7.
Ali, S.,
S. J. Mustafa,
and
W. J. Metzger.
Adenosine receptor-mediated bronchoconstriction and bronchial hyperresponsiveness in allergic rabbit model.
Am. J. Physiol.
266 (Lung Cell. Mol. Physiol. 10):
L271-L277,
1994[Abstract/Free Full Text].
8.
Biden, T. J.,
and
C. B. Wallhem.
Calcium regulates the inositol tris/tetrakisphosphate pathway in intact and broken preparations of insulin-secreting RINmSF cells.
J. Biol. Chem.
261:
11931-11939,
1986[Abstract/Free Full Text].
9.
Bjorck, T.,
L. E. Gustafsson,
and
S. E. Dahlen.
Isolated bronchi from asthmatics are hyperresponsive to adenosine which apparently acts indirectly by liberation of leukotrienes and histamine.
Am. Rev. Respir. Dis.
145:
1087-1091,
1992[Medline].
10.
Bleasdale, J. E.,
N. R. Fitzpatrick,
R. J. Smith,
and
S. Bunting.
Selective inhibition of receptor-coupled phospholipase C-dependent process in human platelets and polymorphonuclear neutrophils.
J. Pharmacol. Exp. Ther.
255:
756-768,
1990[Abstract].
11.
Cauvin, C.,
R. Loutenhiser,
and
C. Van Breemen.
Mechanism of calcium antagonist-induced vasodilation.
Annu. Rev. Pharmacol. Toxicol.
23:
373-396,
1983[Medline].
12.
Chan-Yeung, M.,
and
J. L. Malo.
Aetiological agents in occupational asthma.
Eur. Respir. J.
7:
346-371,
1994[Abstract/Free Full Text].
13.
Chilvers, E. R.,
B. J. Lynch,
and
R. A. T. Challiss.
Phosphoinositide metabolism in airway smooth muscle.
Pharmacol. Ther.
62:
221-245,
1994[Medline].
14.
Cox, D. A.,
S. W. Watts,
and
M. L. Cohen.
Neomycin selectively inhibits 5-hydroxytryptamine-induced contraction in the guinea pig trachea.
J. Pharmacol. Exp. Ther.
277:
954-959,
1996[Abstract].
15.
Cushing, D. J.,
S. R. Makujina,
M. H. Sabouni,
and
S. J. Mustafa.
Protein kinase C and phospholipase C in adenosine receptor-mediated relaxation in coronary artery.
Am. J. Physiol.
261 (Heart Circ. Physiol. 30):
H1848-H1854,
1991[Abstract/Free Full Text].
16.
Cushley, M. J.,
A. E. Tattersfield,
and
S. T. Holgate.
Effect of inhaled adenosine and guanosine on the airway resistance in normal and asthmatic subjects.
Br. J. Clin. Pharmacol.
15:
161-165,
1983[Medline].
17.
Driver, A. G.,
C. A. Kukoly,
S. Ali,
and
S. J. Mustafa.
Adenosine in bronchoalveolar lavage fluid in asthma.
Am. Rev. Respir. Dis.
148:
91-97,
1993[Medline].
18.
El-Hashim, A.,
B. D'Agostino,
M. G. Matera,
and
C. Page.
Characterization of adenosine receptor involved in adenosine-induced bronchoconstriction in allergic rabbits.
Br. J. Pharmacol.
119:
1262-1268,
1996[Abstract].
19.
Gerwins, P.,
and
B. B. Fredholm.
Stimulation of adenosine A1 receptors and bradykinin receptors, which act via different G proteins, synergistically raises inositol 1,4,5-trisphosphate and intracellular free calcium in DDT1 MF-2 smooth muscle cells.
Proc. Natl. Acad. Sci. USA
89:
7330-7334,
1992[Abstract].
20.
Gilman, A. G.
G-proteins: transducers of receptor generated signals.
Annu. Rev. Biochem.
56:
615-649,
1987[Medline].
21.
Irvine, R. F.,
and
R. M. Moor.
Inositol (1,3,4,5)tetrakisphosphate-induced activation of sea urchin eggs requires the presence of inositol trisphosphate.
Biochem. Biophys. Res. Commun.
146:
284-290,
1987[Medline].
22.
Jones, A. W.,
B. B. Geisbuhler,
S. D. Shukla,
and
J. M. Smith.
Altered biochemical and functional responses in aorta from hypertensive rats.
Hypertension
11:
627-634,
1988[Abstract].
23.
Joshi, S.,
W. Abebe,
and
D. K. Agrawal.
G-proteins in guinea pig airway smooth muscle: identification and functional involvement.
Pharmacol. Res.
33:
195-202,
1996[Medline].
24.
Langlands, T. M.,
I. M. Rodger,
and
J. Diamond.
The effect of M & B 22948 on metacholine and histamine-induced contraction and inositol 1,4,5-trisphosphate levels in guinea-pig tracheal tissue.
Br. J. Pharmacol.
98:
336-338,
1989[Abstract].
25.
Metzger, W. J.
Late phase asthmatic responses in the allergic rabbit.
In: Late Phase Allergic Reactions, edited by W. Dorsch. Boca Raton, FL: CRC, 1990, p. 347-362.
26.
National, Heart, Lung, and Blood Institute.
Facts About Asthma. Bethesda, MD: NHLBI, 1990, p. 1-7. (NIH Publ. 90-2339)
27.
Nichols, A. J.,
and
R. R. Ruffolo.
The relationship of alpha-adrenoceptor reserve and agonist intrinsic efficacy to calcium utilization in the vasculature.
Trends Pharmacol. Sci.
9:
236-241,
1988[Medline].
28.
Odeagbo, A. S. O.,
and
K. U. Malik.
Mechanism of vascular actions of prostacycline in the rat isolated perfused mesenteric arteries.
J. Pharmacol. Exp. Ther.
252:
26-34,
1990[Abstract].
29.
Phillippe, M.
Neomycin inhibition of hormone-stimulated smooth muscle contractions in myometrial tissue.
Biochem. Biophys. Res. Commun.
205:
245-250,
1994[Medline].
30.
Ranhosky, A.,
J. Kempthorne-Rawson,
and
the Intravenous Dipridamole Thallium Imaging Study Group.
The safety of intravenous dipyridamole thallium myocardial perfusion imaging.
Circulation
81:
1205-1209,
1990[Abstract].
31.
Sabouni, M. H.,
T. Hussain,
D. J. Cushing,
and
S. J. Mustafa.
G proteins subserve relaxations mediated by adenosine receptors in human coronary artery.
J. Cardiovasc. Pharmacol.
18:
696-702,
1991[Medline].
32.
Salari, H.,
A. Bramley,
J. Langlands,
S. Howard,
M. C. Young,
H. Chan,
and
R. Schellenbery.
Effect of phospholipase C inhibitor U-73122 on antigen-induced airway smooth muscle contraction in guinea pigs.
Am. J. Respir. Cell Mol. Biol.
9:
405-410,
1993[Medline].
33.
Schutze, S.,
K. Potthoff,
T. Machleidt,
D. Berkovic,
K. Wiegmann,
and
M. Kronke.
TNF activates NF-kappa B by phosphatidylcholine-specific phospholipase C-induced "acidic" sphingomyelin breakdown.
Cell
71:
765-776,
1992[Medline].
34.
Siess, W.,
and
E. G. Lapetina.
Neomycin inhibits inositol phosphate formation in human platelets stimulated by thrombin but not other agonists.
FEBS Lett.
201:
53-57,
1986.
35.
Smith, R. T.,
L. M. Sam,
T. M. Tusten,
G. L. Bundy,
G. G. Bala,
and
T. E. Bleasdale.
Receptor-coupled signal transduction in human polymorphonuclear neutrophils. Effects of novel inhibitor of phospholipase C-dependent process on cell responsiveness.
J. Pharmacol. Exp. Ther.
253:
688-697,
1990[Abstract].
36.
Stahl, M. L.,
C. R. Ferenz,
K. L. Kelleher,
R. W. King,
and
J. L. Knoff.
Sequence similarities of phospholipase C with the non-catalytic region of src.
Nature
322:
269-272,
1988.
37.
Suh, P. G.,
S. H. Ryer,
W. C. Choi,
K. Y. Lee,
and
S. G. Rhee.
Monoclonal antibodies to three phospholipase C isozymes from bovine brain.
J. Biol. Chem.
263:
14497-14504,
1988[Abstract/Free Full Text].
38.
Thompson, A. K.,
S. P. Mostafapour,
L. C. Delinger,
J. E. Bleasdale,
and
S. K. Fisher.
The aminosteroid U-73122 inhibits muscarinic receptor sequestration and phosphoinositide hydrolysis in SK-N-SH neuroblastoma cells. A role for Gp in receptor compartmentation.
J. Biol. Chem.
266:
23856-23862,
1991[Abstract/Free Full Text].
39.
Turla, M. B.,
and
C. Webb.
Augmented phosphoinositide metabolism in aortas from genetically hypertensive rats.
Am. J. Physiol.
258 (Heart Circ. Physiol. 27):
H173-H178,
1990[Abstract/Free Full Text].
Am J Physiol Lung Cell Mol Physiol 275(5):L990-L997
0002-9513/98 $5.00
Copyright © 1998 the American Physiological Society