Department of Physiology, Northeastern Ohio Universities College of Medicine, Rootstown, Ohio 44272-0095
Submitted 10 October 2002 ; accepted in final form 14 May 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
pulmonary edema; -adrenergic receptor signaling pathway; receptor desensitization; alveolar epithelial type II cells; adenylyl cyclase; adenosine 3',5'-cyclic monophosphate; protein kinase A
This functional impairment was accompanied by a reduction in the number of
total -ARs on alveolar epithelial type II (ATII) cells freshly isolated
from rats infused with Iso
(14), but a lack of
correlation between the ability of terbutaline to stimulate ALC and the
-AR density suggested that additional desensitization mechanisms must
have played a role in producing the dose-dependent ALC responses. Although
significant attention has been focused on examining desensitization events at
the
-AR and G protein levels, there is emerging evidence that
desensitization may be a phenomenon that affects additional downstream points
in the
-AR signaling pathway. For example, McMartin and Summers
(13) recently reported that
left atrial tissue and left and right ventricular papillary muscles obtained
from rats infused with Iso for 14 days exhibited an impaired ability to
contract in response to both forskolin (a direct activator of adenylyl
cyclase) and dibutyryl-cAMP (a stable cAMP analog). Accordingly, the objective
of this study was to determine if additional impairments in the lung
-AR
signaling pathway developed downstream of the
-AR in rats infused with
Iso for 48 h. To do so, we examined the ability of forskolin to increase ALC
and to stimulate cAMP production in ATII cells isolated from rats infused with
Iso. We also evaluated the abilities of a stable cAMP analog to increase ALC
and of cAMP to increase protein kinase A (PKA) activity in ATII cells isolated
from rats infused with Iso.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Male Sprague-Dawley rats (n = 96) weighing 250-300 g (Harlan, Chicago, IL) were used in this study. The rats were housed in the Comparative Medicine Unit at the Northeastern Ohio Universities College of Medicine for at least 1 wk under temperature-controlled conditions [20 ± 2°C (mean ± SD)] and at a relative humidity of 50 ± 10% before experimental use. The rats were fed a standard rat chow and had water ad libitum. All experiments were approved by the Northeastern Ohio Universities College of Medicine Institutional Animal Care and Use Committee.
Miniosmotic pumps (Alzet model 2001; Durect, Cupertino, CA) were filled
under sterile conditions with either the -AR agonist (-)-isoproterenol
(+)-bitartrate (Iso; Sigma Chemical, St. Louis, MO) or its vehicle (0.001 N
HCl) and primed in sterile saline at 37°C overnight. The next morning, the
filled pumps were aseptically implanted subcutaneously under halothane
anesthesia as previously described
(14). Iso concentrations were
selected to allow the pumps to deliver the drug at infusion rates of 4, 40, or
400 µg Iso base · kg-1 ·
h-1. These Iso administration rates were selected
because they represent the range of infusion rates that have been used in
previous studies to evaluate the ability of Iso to produce
-AR
downregulation (8,
14,
24,
25).
Determination of ALC
ALC was measured as previously described
(3,
14). The rats were
anesthetized with 80 mg/kg ip pentobarbital sodium (Abbot Laboratories, N.
Chicago, IL), with the anesthetic being supplemented as needed. Body
temperature was monitored using a rectal temperature probe and was maintained
using a water-perfused heating pad. A polyethylene tracheal cannula (PE-240;
Clay Adams, Becton-Dickinson, Sparks, MD) was placed in the rat's airway via a
tracheotomy and connected to a mechanical ventilator (Harvard Apparatus,
Nantucket, MA). The lungs were ventilated with a
FIO2 = 1.0 at a respiratory rate of 40
breaths/min with an average tidal volume of 2.6 ± 0.2 (SD) ml. Peak
inspiratory pressure was 9.2 ± 1.2 Torr under baseline conditions, and
endexpiratory pressure was atmospheric. The rat was placed at a 45° angle
(head elevated), and a polyethylene catheter (PE-50; Clay Adams) was inserted
through a port in the tracheal cannula and in the lungs for liquid
instillation. The rats were allowed to stabilize for 10 min after surgery
before starting the experiment. At this time, 3 ml/kg of a 5% BSA (Sigma)
solution in Ringer lactate (Baxter Healthcare, Deerfield, IL) was instilled in
the left lung at a rate of 0.20 ml/1.5 min using a 1-ml syringe. The solution
was previously adjusted with NaCl to an osmolality of 315 mM/kg. The alveolar
instillate was left in the lungs for 1 h beginning at the completion of
instillation. After 1 h, a thoracotomy was done. A blood sample was obtained
for blood gas analysis via aortic puncture and analyzed using a Radiometer
system. Average blood gas determinations were as follows:
PO2: 360 ± 162 (SD) Torr,
PCO2: 37.0 ± 8.8 Torr, pH: 7.43 ± 0.07.
The rat was killed by exsanguination, the lungs were removed, and a sample of
the remaining instilled liquid was aspirated for analysis of albumin
concentration by refractometry. The refractometer (American Optical, Buffalo,
NY) was calibrated using a series of albumin standards (Sigma). ALC was
determined using the following mass balance equation
![]() | (1) |
ATII Cell Isolation
The ATII cells were isolated using the technique of Dobbs et al. (4). Briefly, the rats were anesthetized with pentobarbital sodium (80 mg/kg ip) and heparinized (1,000 units). A tracheotomy was done, and an 18-gauge angiocatheter (Becton-Dickinson Infusion Therapy Systems, Sandy, UT) was inserted. The chest was opened, and the lungs were removed. The lungs were perfused through the pulmonary artery to remove blood and lavaged to remove alveolar macrophages. The lungs were then digested with 20 ml elastase (3 U/ml; Worthington Biochemical, Lakewood, NJ) for 20 min at 37°C. The digested tissue was minced in the presence of FBS (Hyclone, Logan, UT) and DNase (Sigma) and filtered through sterile gauze and then through 70-µm nylon mesh filters (Becton-Dickinson Labware, Franklin Lakes, NJ). The filtrate was centrifuged at 400 g, and the cell pellet was resuspended in DMEM (Irvine Scientific, Santa Ana, CA) containing an antibiotic-antimycotic cocktail of penicillin G, streptomycin sulfate, and amphotericin B (GIBCO-BRL, Grand Island, NY) and glutamine (Irvine Scientific). The type II cells were purified by differential adherence to IgG (Sigma)-coated plates. Cell yield and purity (85%) were determined by using a Beckman Coulter Z1 Coulter particle counter and by tannic acid staining (11).
Experimental Design
Effect of prolonged Iso infusion on the ability of forskolin to
increase ALC. In this set of experiments, we determined if 48 h of Iso
infusion (at rates of either 4, 40, or 400 µg ·
kg-1 · h-1, n = 6
for each infusion rate) affected the ability of the adenylyl cyclase
activator, forskolin, to increase ALC. After the 48-h Iso infusion period, the
rats were anesthetized with pentobarbital sodium, and an external jugular vein
was cannulated. An intravenous forskolin (Sigma) infusion was started (16.6
µg · kg-1 · min-1
in a volume flow of 0.026 ml/min) and maintained for the duration of the
experiment. Forskolin was dissolved in 5% DMSO (Sigma) in saline. The 5% BSA
solution, containing 10-4 M dl-propranolol (Sigma), was
instilled in the lung for measurement of ALC 15 min after starting the
forskolin infusion. These experiments were done in the presence of propranolol
to ensure that the forskolin-stimulated increase in ALC was not mediated by
-AR activation. Low forskolin concentrations have been shown to
potentiate accumulation of cAMP produced by a variety of agents, including
adrenergic agonists (22). ALC
was also measured in vehicle-infused rats administered forskolin and
propranolol (n = 12) and those treated with 5% DMSO (forskolin
vehicle) and propranolol (n = 6).
Effect of prolonged Iso infusion on the ability of forskolin to increase cAMP concentration in isolated ATII cells. For these experiments, rats were infused with Iso (at rates of 4, 40, or 400 µg · kg-1 · h-1) or vehicle (0.001 N HCl) for 48 h. After 48 h, the animals were heparinized and anesthetized as described in ATII Cell Isolation, and the lungs were removed for isolation of ATII cells. A sufficient number of ATII cells could be isolated from the lungs of each rat to allow a complete cAMP analysis for each animal. Four rats were used for each infusion rate.
Immediately after isolation, the cells were assayed for cAMP concentrations, as previously described (16) using a commercially available enzyme immunoassay (EIA) kit (Direct cAMP Correlate-EIA; Assay Designs, Ann Arbor, MI). We used the same number of cells (1.5 x 106) for each assay, thus ensuring that the cell number would not be a variable. cAMP was always measured in the presence of the phosphodiesterase inhibitor IBMX (10-3 M; Sigma) at 37°C under baseline conditions and after a 10-min forskolin stimulation (10-4 M). Additional cAMP determinations were also made at 4°C and in the presence of 1% DMSO (forskolin vehicle). DMSO was found to have no effect on cAMP production. The EIA had a sensitivity of 0.39 pmol/ml and intra- and interassay variabilities of 8.9 and 13.1%, respectively.
Effect of prolonged Iso infusion on the ability of 8-bromoadenosine-3',5'-cyclic monophosphorothioate, Sp-isomer to increase ALC. In this set of experiments, we determined if a prolonged Iso infusion (at rates of either 4, 40, or 400 µg · kg-1 · h-1, n = 6-7) to rats affected the ability of the stable cAMP-dependent protein kinase activator 8-bromoadenosine-3',5'-cyclic monophosphorothioate, Sp-isomer (Sp-8-bromo-cAMPS; 10-4 M dissolved in the instillate; Biolog Life Science Institute, Bremen, Germany), to increase ALC. ALC was also measured under baseline conditions (n = 7) and after Sp-8-bromo-cAMPS stimulation (n = 6) in groups of rats receiving a 48-h vehicle infusion.
Effect of prolonged Iso infusion on the ability of cAMP to stimulate
PKA activity in isolated ATII cells. Rats were infused with Iso (at rates
of 4, 40, or 400 µg · kg-1 ·
h-1) or vehicle (0.001 N HCl) for 48 h (n = 3
for each infusion rate). After 48 h, ATII cells were isolated as described in
ATII Cell Isolation. A sufficient number of ATII cells could be
isolated from the lungs of each rat to allow an analysis of baseline and
cAMP-stimulated PKA activity in each animal. PKA activity was determined in
the freshly isolated ATII cells using a commercially available ELISA
(Calbiochem, San Diego, CA) that uses a synthetic PKA pseudosubstrate peptide
and a monoclonal antibody that recognizes only the phosphorylated form of the
pseudosubstrate peptide. Briefly, 4.0 x 107 cells were
used for the assay. The cells were washed in ice-cold DMEM and pelleted at
11,600 g, and the supernatant was removed. The cell pellet was then
snap-frozen in liquid nitrogen and stored at -80°C until the assay was
run. On the day of assay, the cell pellet was resuspended in 1 ml ice-cold
sample preparation buffer according to the protocol of the assay kit. The cell
membranes were disrupted by sonication five times for 10 s each. The cell
suspension was then centrifuged at 100,000 g for 1 h at +4°C, and
the supernatant was collected. The assay was then immediately run according to
the kit protocol.
Statistical Analysis
Multigroup comparisons of data were done by ANOVA followed by the Student Newman-Keul's post hoc test. Comparisons between two groups were made by unpaired Student's t-test.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The pattern of weight changes observed in the rats of this study after 48 h of vehicle or Iso infusion was similar to that observed in our previous study (14). Rats infused with vehicle or Iso at 4 µg · kg-1 · h-1 exhibited, respectively, 3.9 ± 0.3% and 6.6 ± 0.4% (means ± SE) weight gains during the 48-h period after pump implantation. In contrast, rats infused with Iso at rates of 40 and 400 µg · kg-1 · h-1 exhibited, respectively, a weight increase of 1.4 ± 0.6% or a weight loss of 6.1 ± 0.8% during the 48-h period.
Effects of Prolonged Iso Infusion on the Ability of Forskolin to Increase ALC and cAMP Concentration in Isolated ATII Cells
The average baseline ALC (in the presence of propranolol) observed in vehicle-infused (0 µg · kg-1 · h-1 Iso) rats was 16.6 ± 1.7% (mean ± SE) of the instilled volume absorbed per hour. ALC in vehicle-infused rats administered forskolin and propranolol was 101% greater (33.4 ± 2.1%, P < 0.05, Fig. 1A) than that observed under baseline conditions. In rats infused with Iso at the 40 and 400 µg · kg-1 · h-1 rates, forskolin-stimulated ALC was reduced by 25 and 38%, respectively (both P < 0.05, Fig. 1A). ALC measured after forskolin administration (20.7 ± 2.0%) in the 400 µg · kg-1 · h-1 Iso infusion group was not different from that observed in the vehicle-infused rats under baseline conditions.
|
Although the ability of forskolin to stimulate ALC was reduced in rats infused with Iso at the 40 µg · kg-1 · h-1 infusion rate, an impairment in ATII cell forskolin-stimulated cAMP production (70%, P < 0.05) was only observed at the highest Iso infusion rate (Fig. 1B).
Effects of Prolonged Iso Infusion on the Ability of Sp-8-bromo-cAMPS to Increase ALC and of cAMP to Stimulate PKA Activity in Isolated ATII Cells
The average baseline ALC observed in vehicle-infused rats was 22.0 ± 1.4% (mean ± SE) of the instilled volume absorbed per hour. ALC in vehicle-infused (0 µg · kg-1 · h-1 Iso in Fig. 2) rats administered Sp-8-bromo-cAMPS was 121% higher (48.7 ± 3.0%, P < 0.05) than that observed under baseline conditions. In rats infused with Iso at 40 and 400 µg · kg-1 · h-1, Sp-8-bromo-cAMPS-stimulated ALC was reduced by 25 and 51%, respectively (both P < 0.05, Fig. 2) compared with Sp-8-bromo-cAMPS-stimulated ALC in vehicle-infused rats. ALC measured after Sp-8-bromo-cAMPS administration in the 400 µg · kg-1 · h-1 Iso infusion group (23.8 ± 1.2%) was not different from that observed in the vehicle-infused rats under baseline conditions. ALCs observed in the 4, 40, and 400 µg · kg-1 · h-1 groups were all significantly different (P < 0.05) from one another.
|
Basal PKA activity of freshly isolated ATII cells from rats infused with any of the three Iso infusion rates did not differ from that observed in ATII cells obtained from vehicle-infused rats (Fig. 3). In ATII cells harvested from vehicle-infused rats, cAMP stimulated the PKA activity by 387% (Fig. 3). Iso infusion inhibited the ability of cAMP to stimulate PKA activity in a dose-dependent manner, with no stimulation being observed at the 400 µg · kg-1 · h-1 infusion rate (Fig. 3).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In initial experiments, we administered the adenylyl cyclase activator
forskolin to rats infused with Iso and observed that the ability of forskolin
to increase ALC was reduced in a dose-dependent pattern
(Fig. 1A) that was
reminiscent of that previously observed with -AR agonists
(14). Because forskolin can
potentiate the action of
-AR agonists
(22), we coadministered
propranolol with forskolin in these experiments to ensure that the action of
forskolin was not the result of
-AR activation by endogenous
catecholamines or Iso. Because we were able to eliminate this as a
possibility, the results of these experiments demonstrate the existence of at
least one additional site of desensitization in the
-AR signaling
pathway downstream of the
-AR.
Although forskolin is a direct adenylyl cyclase activator, these
observations cannot in themselves be interpreted to mean that the site of
desensitization is at the level of adenylyl cyclase. A similar pattern of ALC
responses to forskolin could have also been obtained if the desensitization
site was located at some point downstream of this enzyme. To answer this
question, we conducted additional experiments in which we evaluated the
ability of forskolin to increase cAMP in ATII cells freshly isolated from rats
infused with Iso and found that forskolin-stimulated cAMP production was
impaired in ATII cells obtained from rats that had received the highest Iso
(400 µg · kg-1 ·
h-1) infusion rate
(Fig. 1B), but not at
the lower Iso infusion rates. Although there is evidence that prolonged
-AR stimulation may lead to increased phosphodiesterase activity in
whole lung tissue (5), which in
turn might decrease cAMP accumulation, this seemed unlikely since our
experiments were conducted in the presence of relatively high concentrations
of the phosphodiesterase inhibitor IBMX. These data thus indicate that the
impaired forskolin-stimulated cAMP accumulation was the result of a decreased
cAMP production rather than an enhanced degradation. The mechanism responsible
for this impairment is unknown but is presumably the result of a reduction in
adenylyl cyclase expression or activity. With respect to activity, adenylyl
cyclase is capable of receiving and integrating both stimulatory and
inhibitory inputs from a variety of sources, with the nine known mammalian
isoforms exhibiting variable responsiveness to different stimuli
(23). It is thus possible that
prolonged Iso infusion may have altered the balance of afferent stimulatory
and inhibitory signaling to adenylyl cyclase within the ATII cell.
These observations suggest that a limitation in the ATII cell's ability to
produce cAMP could have contributed to the impaired ability of forskolin (as
well as -AR agonists) to increase ALC in rats infused with the highest
Iso infusion rate (400 µg · kg-1 ·
h-1). In contrast, the ability of forskolin to stimulate
ALC in vivo was reduced at the 40 µg · kg-1
· h-1 Iso infusion rate
(Fig. 1A), but there
was no accompanying impairment in the ability of forskolin to increase cAMP in
the isolated ATII cells (Fig.
1B). This disparity suggests that the reduced ability of
forskolin to increase ALC in rats infused with Iso at 40 µg ·
kg-1 · h-1 (and perhaps at
higher Iso infusion rates as well) resulted from additional desensitization
events occurring downstream of the point of cAMP production.
To investigate this latter possibility, we administered the stable cAMP
analog and PKA activator Sp-8-bromo-cAMPS to rats infused with Iso and
observed that the ability of Sp-8-bromo-cAMPS to increase ALC was decreased in
rats infused with 40 and 400 µg · kg-1
· h-1 Iso
(Fig. 2). These data indicate
that prolonged Iso infusion also interferes with alveolar epithelial -AR
signaling at some site in the signaling pathway at the level of PKA or beyond.
This site could represent PKA itself, the membrane Na+ transport
proteins (i.e., apical Na+ channels or
Na+-K+-ATPase), and/or some intermediate step.
To determine if PKA became desensitized, we evaluated the ability of cAMP
to stimulate PKA activity in ATII cells isolated from rats infused with
vehicle or Iso. We observed that Iso infusion resulted in a dose-dependent
impairment in the ability of cAMP to stimulate PKA activity, with a total loss
of PKA stimulation at the 400 µg · kg-1
· h-1 Iso infusion rate
(Fig. 3). These observations
suggest that the Iso-induced impaired ability of Sp-8-bromo-cAMPS to increase
ALC resulted from desensitization of PKA. It is important to point out,
however, that this conclusion is predicated on the assumption of an intact
transalveolar epithelial Na+ transport ability. In this regard, the
level of PKA stimulation (or any other component of the -AR signaling
pathway) would be of little consequence if Iso infusion impaired the function
of the alveolar epithelial Na+ transport pathways.
The combined results of our previous
(14) and current studies show
that by 48 h of 400 µg · kg-1 ·
h-1 Iso infusion, alterations in the ATII cell -AR
signaling pathway develop at the receptor, adenylyl cyclase, and PKA levels.
The inability of Sp-8-bromo-cAMPS to increase ALC and of cAMP to stimulate PKA
activity that developed at this infusion rate suggests that an important
rate-limiting step in the ability of
-AR agonists to increase ALC
develops at the PKA level. Our data do not provide insight, however, about the
physiological importance of the receptor downregulation and impaired adenylyl
cyclase activity, because it is not known if these changes by themselves are
of sufficient magnitude to impair signaling. It is thus not clear if the
overall impairment in
-AR agonist-stimulated ALC reflects the
contribution of the effects of multiple desensitization events or of a single
rate-limiting step residing at the level of PKA.
The development of downstream -AR signaling pathway defects after
prolonged
-AR agonist exposure has been found to occur in other tissues
and cell types and is consistent with our observations. For example, a number
of studies have reported the development of impaired cardiac
-AR
signaling downstream of the
-AR in animals and cultured heart cells
administered
-AR agonists for prolonged time periods
(9,
13,
18,
19) as well as in myocytes
obtained from patients with congestive heart disease
(7). These signaling defects
include impaired forskolin- and stable cAMP-stimulated contractile responses
and reduced forskolin-stimulated cAMP production. Additionally, exposure of a
variety of cultured cell types to cAMP for prolonged periods produces
desensitization of PKA mediated by a variety of mechanisms
(10,
17,
20,
26). The results of this study
extend this concept to the alveolar epithelium and indicate that the
development of such impairments in postreceptor signaling can have functional
significance.
The ability of -AR agonists to increase ALC suggests that
-AR
agonist therapy might be used clinically for the treatment of alveolar edema
(1,
6). The combined results of the
current and our previous study
(14) indicate that prolonged
Iso infusion results in impaired signaling at multiple sites in the alveolar
epithelial
-AR signaling pathway, which exhibit differing
desensitization thresholds. For example,
-AR downregulation and impaired
PKA stimulation were apparent at the 40 µg ·
kg-1 · h-1 infusion rate,
whereas an impaired adenylyl cyclase function did not develop until the Iso
infusion rate was increased to 400 µg · kg-1
· h-1. These observations suggest that attention
might need to be given to tailoring
-AR agonist therapy to maximize the
increase in ALC while minimizing the potential for desensitization. Sartori et
al. (21) have recently
reported that mice infused with the specific
2-AR partial
agonist albuterol for up to 6 days exhibited a reduction in
-AR density
and an impaired
-AR agonist-stimulated cAMP accumulation in peripheral
lung tissue but still retained a maximum ability to increase ALC in response
to terbutaline administration. These data suggest that the mice did not
undergo PKA desensitization. The reason for these differences is unclear but
could relate to species differences, agonist characteristics, or drug doses.
An answer to this question may provide additional important insight into the
potential efficacy of
-AR agonist therapy for treatment of severe
pulmonary edema.
In summary, we previously identified -AR downregulation as a
potential mechanism that may have played a role in producing the impaired
ability of
-AR agonists to increase ALC observed in rats after 48 h of
Iso infusion. The results of the current study extend this observation by
showing that additional sites of the
-AR signaling pathway undergo
desensitization as well at clinically relevant agonist doses. These sites
include the enzymes adenylyl cyclase and PKA, with the latter being a
potential rate-limiting step.
![]() |
DISCLOSURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
FOOTNOTES |
---|
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. Section 1734 solely to indicate this fact.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|