Thermal acclimation of surfactant secretion and its regulation by adrenergic and cholinergic agonists in type II cells isolated from warm-active and torpid golden-mantled ground squirrels, Spermophilus lateralis
1 Environmental Biology, School of Earth and Environmental Sciences,
University of Adelaide, Adelaide SA 5005, Australia
2 Department of Zoology, University of British Columbia, Vancouver BC
V6T1Z4, Canada
* Author for correspondence (e-mail: sandra.orgeig{at}adelaide.edu.au)
Accepted 28 May 2003
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Summary |
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Key words: ground squirrel, Spermophilus lateralis, pulmonary surfactant, hibernation, torpor, alveolar type II cell, cholinergic, adrenergic
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Introduction |
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Homeothermic mammals, such as humans and rats, experience surfactant
dysfunction and respiratory distress with small fluctuations in body
temperature (Inoue et al.,
1981; Meban, 1978
;
Peterson and Davis, 1986
).
However, heterothermic mammals such as fat-tailed dunnarts Sminthopsis
crassicaudata and golden-mantled ground squirrels Spermophilus
lateralis regularly experience rapid changes in body temperature when
they enter a depressed metabolic state, known as torpor or hibernation,
respectively (Geiser and Ruf,
1995
). Temperature-induced changes in surfactant amount and/or
composition have been observed during the stress-induced torpor of fat-tailed
dunnarts S. crassicaudata
(Langman et al., 1996
) and the
daily torpor of the microchiropteran bats Chalinolobus gouldii
(Codd et al., 2000b
) and
Nyctophilus geoffroyi (Slocombe
et al., 2000
). In dunnarts, after 8 h of torpor, there are
increases in the relative amounts of PL, disaturated phospholipid (DSP) and
cholesterol (CHOL) (Langman et al.,
1996
). These changes correlate with changes in surface activity
and, therefore, enable dunnart surfactant to function effectively at torpid
body temperatures (Lopatko et al.,
1998
). Similarly, total PL increased in lavage fluid collected
from mildly cold-acclimated ground squirrels Spermophilus richardsoni
(Melling and Keough, 1981
). In
contrast, surfactant PL decreased or did not change during torpor in the
microchiropteran bats N. geoffroyi and C. gouldii,
respectively (Codd et al.,
2000b
; Slocombe et al.,
2000
). The different responses of bats, dunnarts and squirrels
probably reflect the different physiological states of the animals during
torpor (Codd et al.,
2000a
).
During hibernation, ground squirrels enter a much deeper and more prolonged
torpor than that reported for the stress-induced torpor of small marsupials
and daily torpor of bats (Geiser and Ruf,
1995). The depth and duration of their torpor bouts makes
squirrels an excellent model for studying the thermal dynamics of a mammalian
surfactant system. Furthermore, ground squirrels enter hibernation readily
under appropriate laboratory conditions. However, very little is known about
the effects of torpor on the surfactant system in hibernators. During the
hibernating season, golden-mantled ground squirrels Spermophilus
lateralis are capable of reducing their body temperatures to as low as
0-5°C during torpor, and torpor bouts usually last for 10-14 days at a
time (Milsom et al., 1999
).
After a torpor bout, the ground squirrels will spontaneously arouse,
increasing their body temperature to 37°C for a brief period (h) before
returning to a torpid state. Although hibernation is highly advantageous in
terms of energy conservation, the long duration and depth of torpor bouts
experienced by ground squirrels are likely to have marked effects on the
composition, function and regulation of the surfactant system. Alternatively,
given the different type of torpor and the annual regularity of the
hibernating season, ground squirrels may have adopted novel and unique
approaches for maintaining surfactant function at both warm-active (37°C)
and torpid (0-5°C) body temperatures.
The low temperatures experienced by ground squirrels during torpor bouts
are likely to also have a profound effect on the release of surfactant into
the lung and the regulatory and secretory pathways controlling surfactant
release. High temperatures increase metabolic rate and thus, may directly
stimulate the rate of synthesis and/or secretion of lamellar bodies from type
II cells (Chander and Fisher,
1990). Conversely, low temperatures decrease metabolic rate, and
may therefore lower the rate of surfactant secretion. In ATII cells isolated
from homeothermic rats, a decrease in incubation temperature to 5°C
virtually abolishes surfactant secretion
(Dobbs and Mason, 1979
). Basal
secretion is also significantly lower in type II cells isolated from
warm-active dunnarts when incubated at 15°C compared to 37°C
(Ormond et al., 2001
). In
dunnart type II cells, however, the decrease in the rate of surfactant
secretion has a Q10 value of 1.3
(Ormond et al., 2001
). The
fact that this Q10 value is lower than 2, indicates that the
secretory pathway in dunnart type II cells is relatively insensitive to
temperature and must be regulated or altered in some way to counteract the
kinetic effects of decreasing temperature
(Ormond et al., 2001
;
Schmidt-Nielson, 1997
).
Therefore, we suggest that in ground squirrels, the composition, function and
cellular biomechanics of the surfactant system must also be modified to enable
efficient functioning at body temperatures of 0-5°C.
The sympathetic nervous system is an important regulator of surfactant
release in mammals (Chander and Fisher,
1990). Adrenergic factors are released from the sympathetic
nervous system (SNS) and bind to membrane-bound ß-adrenergic receptors on
type II cells to activate the signalling molecule, cAMP, and enhance
surfactant secretion (Brown and Longmore,
1981
; Dobbs and Mason,
1978
; Wood et al.,
1997
). Adrenergic agonists stimulate surfactant secretion in type
II cells isolated from homeothermic animals such as rat
(Brown and Longmore, 1981
;
Chander and Fisher, 1990
),
chicken (Sullivan and Orgeig,
2001
) and tammar wallaby
(Miller et al., 2001
),
heterothermic animals such as fat-tailed dunnart
(Ormond et al., 2001
), and
ectothermic animals such as bearded dragon, frog, lungfish
(Wood et al., 2000
) and
crocodile (Sullivan et al.,
2002
). In type II cells isolated from bearded dragons and
fat-tailed dunnarts, this response to isoproterenol did not change regardless
of assay temperature (Ormond et al.,
2001
; Wood et al.,
1999
,
2000
). Thus, the regulation of
surfactant secretion by the ß-adrenergic signalling pathway appears to be
relatively temperature-insensitive in lizards and dunnarts. Sympathetic
output, however, probably decreases markedly during torpor in vivo
(Wood et al., 2000
).
Therefore, regulation by the parasympathetic nervous system (PNS) and
cholinergic agonists, which do not increase metabolic rate, may be more
important in controlling surfactant release during torpor
(Wood et al., 2000
).
Carbamylcholine chloride acts through membrane-bound muscarinic receptors
to increase PC secretion in ATII cells isolated from fat-tailed dunnarts
Sminthopsis crassicaudata (Ormond
et al., 2001; Wood et al.,
1999
,
2000
), and juvenile, unfurred
(heterothermic) tammar wallabies Macropus eugenii
(Miller et al., 2001
). In
direct contrast, type II cells isolated from homeothermic mammals such as
humans and rats do not respond to cholinergic agonists
(Dobbs and Mason, 1979
). This
suggests that heterothermic mammals may have a direct role for the
parasympathetic nervous system in regulating surfactant secretion at low body
temperatures. Type II cells isolated from the ectothermic bearded dragon
Pogona vitticeps (Tb range: 15-40°C;
Tb study=25°C) also respond to carbamylcholine
chloride, but only at a relatively cold assay temperature of 18°C and not
at 37°C (Wood et al.,
1999
). This switch in the response to cholinergic stimulation in
lizard type II cells suggests that the cholinergic signalling pathway is
highly sensitive to temperature changes. However, in fat-tailed dunnarts, the
response of isolated type II cells to carbamylcholine chloride remains the
same, regardless of assay temperature
(Ormond et al., 2001
). This
may enable dunnart type II cells to respond quickly to a physiological change
in autonomic stimulation (adrenergic vs cholinergic) during the rapid
entry and arousal from torpor (Ormond et
al., 2001
). Here, we characterise the effect of temperature on the
control and release of surfactant from squirrel type II cells isolated from
warm-active and torpid ground squirrels. Given the importance of the
surfactant system to lung function, we hypothesize that squirrel type II cells
will retain the ability to secrete surfactant even at very cold body
temperatures. Upregulating their response to adrenergic and cholinergic
stimulation may also increase the sensitivity of type II cells to autonomic
control during torpor. Understanding how the surfactant system can remain
functional over a range of temperatures has important consequences in
hypothermic lung transplantation surgery and in the treatment of hypothermia
and respiratory distress syndromes
(Bernard, 1996
;
Erasmus et al., 1996
;
Osanai et al., 1991
;
Inoue et al., 1981
;
Meban, 1978
).
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Materials and methods |
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Isolation of type II cells
Procedures for isolating type II cells were modified from the methods of
Dobbs et al. (1986a), Wood et
al. (1999
,
2000
) and Ormond et al.
(2001
). Animals were
anaesthetised with an intraperitoneal injection of pentobarbitone sodium
(50-150 mg kg-1 body mass). The trachea was cannulated and the
ground squirrel placed on a ventilator set to a volume of 10 ml and a
frequency of 20-30 breaths min-1 during the perfusion. The thorax
was opened and the lungs were perfused, under gravity at 33 cmH2O
via the pulmonary artery, with a sterile saline solution containing 2
i.u. ml-1 of heparin sodium, until free of blood. The lungs were
lavaged via the tracheal cannula with three separate 10 ml volumes of
ice-cold saline. After lavaging, the lungs were excised from the squirrels,
blotted with sterile gauze to remove excess water and weighed to determine wet
lung mass. The lungs were then placed in a sterile 50 ml tube containing
saline. Aseptic techniques were used from this point in the isolation
procedure and carried out in a laminar flow hood. The lungs were rinsed twice
with sterile phosphate-buffered saline containing 10 U ml-1
penicillin, 10 µg ml-1 streptomycin and 25 ng ml-1
amphotericin and transferred to new 50 ml sterile tubes containing 6 U
ml-1 elastase (120 U per lung), 250 µg ml-1 DNase, 10
U ml-1 penicillin and 10 µg ml-1 streptomycin. The
lungs were shaken continuously at room temperature for 30-40 min. The digested
lungs were further dissociated mechanically by pipetting up and down with a 1
ml pipette for 2 min and filtered through a sterile 100 mesh filter (Sigma
Chemical Company, St Louis, MO, USA) to remove any undigested tissue fragments
and large contaminating cells. Cell suspensions were centrifuged at 200
g for 10 min at 22°C (Beckman GS-6R centrifuge) and the
supernatant discarded.
The cell pellets were resuspended in an appropriate volume of DMEM + 10% foetal bovine serum (FBS) (containing 10 U ml-1 penicillin and 10 µg ml-1 streptomycin) and incubated on tissue culture plates for 1 h at the appropriate temperature (37°C for cells from warm-active squirrels and 4°C for cells from torpid squirrels) to allow any fibroblasts to attach to the plates. The plates were then gently `panned' and rinsed twice with 5 ml of culture medium to remove unattached type II cells. All plate washes were collected and pooled. Cell suspensions were centrifuged at 200 g for 10 min at 22°C (Beckman GS-6R centrifuge) and the supernatant discarded. Sterile bacteriologic plates (size 60/15, Greiner Laboratories, Austria) were coated with 3-5 ml of bovine IgG solution (500 µg ml-1, Sigma Chemical Corp., St Louis, MO, USA) and incubated at 22°C for a minimum of 3 h. IgG-coated plates were then washed twice with 5 ml of phosphate buffered saline (PBS, Sigma) and once with 5 ml of sterile culture medium (DMEM containing 20 mmol l-1 Hepes, 3.7 g l-1 sodium bicarbonate, 100 000 U l-1 penicillin, 100 mg l-1 streptomycin). Cell pellets were resuspended in 4-6 ml per plate of culture medium containing 250 µg ml-1 DNase and added to the bacteriological plates. Plates were incubated at the appropriate temperature (37°C for cells from warm-active squirrels, 4°C for cells from torpid squirrels) with 10% CO2 for 1 h to allow macrophages to attach to the IgG-coated plates. After 1 h, the plates were examined to ensure that macrophages had attached to the plates. The plates were then gently `panned' and rinsed twice with 5 ml of culture medium to remove unattached type II cells. All plate washes were collected and pooled. Final cell suspensions were examined using light and electron microscopy to confirm cell type and purity. Type II cells have a cuboidal shape and contain lamellar bodies and microvilli. The ability to secrete PC also confirmed that these cells were type II epithelial cells. The final cell suspensions isolated by this method were >90% pure type II cells. The remaining proportion of cells consisted of macrophages and neutrophils that had not adhered to the IgG-coated plates. The occasional erythrocyte was also present.
Cell viability was measured by the exclusion of the vital dye, Trypan Blue
(Dobbs et al., 1986b) using a
haemocytometer (Neubauer improved, depth 0.1 mm, 0.0025 mm2).
Viable type II cells actively exclude the Trypan Blue dye and remain clear.
Non-viable type II cells do not exclude the Trypan Blue dye and, therefore,
appear blue. A total of 8 cell counts per cell suspension were performed to
calculate percentage viability. Cell viability was determined using Trypan
Blue on all freshly isolated cells at the time of plating for each experiment.
In addition, cell viability was determined using a lactate dehydrogenase (LDH)
cytotoxicity assay (Roche Diagnostics, GmbH, Germany) after overnight
incubations at each of the temperatures and during the course of the secretion
experiments. The assay was performed as per the manufacturer's
instructions.
Microscopy of squirrel lung
Freshly isolated squirrel lungs were cut into 1 mm3 pieces and
fixed in 4% paraformaldehyde, 1.25% glutaraldehyde, 4% sucrose in 0.1 mol
l-1 PBS, pH 7.2, at 4°C for 3-7 days. The fixed material was
washed in 0.1 mol l-1 PBS and postfixed in 1% Osmium Tetroxide
overnight. Tissue pieces were then stained en bloc in 1.5% uranyl
acetate, dehydrated in 70, 80, 90 and 100% ice-cold acetone, embedded in
Araldite resin and polymerized. Cut sections were mounted on grids and
photographed using a transmission electron microscope (Philips CM 100
TEM).
Measurement of PC secretion
Fresh cell isolates from squirrels were centrifuged at 200
g for 10 min at 22°C (Beckman GS-6R centrifuge) and the
cell pellet resuspended in culture medium containing 10% FBS (heat
inactivated) to give a concentration of 3x106 cells
ml-1. 2 µl ml-1 of [methyl-3H]choline
chloride (specific activity 3.00 Tbq mmol-1, 81.0 Ci
mmol-1, 1 µCi ml-1, Amersham Pharmacia Biotech,
Canada) was added to the final cell suspension. 100 µl of
3H-labelled cell suspension were added to each well of
fibronectin-coated plates to give a density of 3x105 cells
well-1 (0.6x106 cells cm-2).
Fibronectin coated plates (5 µg cm-2) were prepared by adding
100 µl of fibronectin (25 µg ml-1) to each well of a sterile,
flat-bottomed 96-well tissue culture plate (Falcon, Becton Dickinson Labware,
NJ, USA). The plates were incubated at room temperature for 45 min to allow
the fibronectin to bind. The fibronectin solution was removed from the plates
by aspiration, immediately before addition of the cell suspension. Plates were
incubated for 22 h at the appropriate temperature with 10% CO2. The
type II cells attached to the fibronectin-coated plates during this time.
After 22 h, the cells were examined under the microscope to assess
viability and morphology. Cell viability was determined after the 22 h
incubations in wells without radiolabel, using both Trypan Blue and a LDH
cytotoxicity assay, and during the course of the secretion experiments. Stock
solutions (1 mmol l-1) of agonists (adrenergic agonist,
isoproterenol and cholinergic agonist, carbamylcholine chloride) were prepared
immediately prior to their use in sample medium (culture medium containing 1
mmol l-1 sodium ascorbate and 1% FBS) equilibrated to 4°C or
37°C. A total volume of 100 µl, prepared by the addition of 90 µl of
sample medium and 10 µl of 1 mmol l-1 agonist solutions, was
added gently to the experimental wells (three replicates of each). 90 µl of
sample medium, followed by 10 µl of sample medium was added to control
wells. 100 µmol l-1 concentrations of agonists were chosen from
the literature (Brown and Longmore,
1981; Dobbs et al.,
1986a
). Plates were incubated for 30 min and 1 h at either 4°C
or 37°C and in 10% CO2. Following each experiment, the medium
from each well was collected, each well was washed twice with 200 µl of
culture medium and the washes pooled. 100 µl of 0.25% (w/v) trypsin/0.04%
EDTA solution in sterile PBS was added to the wells. Media samples were
centrifuged at 3800 g for 2 min in a capsule microcentrifuge
(Tomy Seiko, Japan). The supernatant was transferred to fresh Eppendorf tubes,
the cell pellet resuspended in 100 µl of fresh culture medium and
centrifuged for a further 2 min to wash any secreted phospholipids from the
cells. This last 100 µl wash was collected and added to the media samples.
Once all the time points had been completed, all plates were incubated at
37°C until the trypsin/EDTA solution had removed all attached cells from
the wells. The trypsin/EDTA solution from each well was added to the cell
pellets (from medium spins) and each well was washed twice with 200 µl of
culture medium and the washes also pooled. Samples were stored at -20°C
until extracted. Lipids were extracted from the media and cell samples using
the method of Bligh and Dyer
(1959
). Unlabelled
L-
-phosphatidylcholine (250 µg) was added as a carrier molecule to
the extractions to improve the recovery of radioactive lipids. Lipids in
chloroform were transferred to 8 ml scintillation vials and evaporated in air
(16 h). Lipids were reconstituted in 2 ml of ReadyOrganicTM liquid
scintillation fluid, vortexed and the radioactivity counted on a liquid
scintillation counter (Beckman LS 3801).
Data analysis
c.p.m. values were obtained for the medium and cell fractions of each
sample. Results are expressed as the percentage secretion of incorporated
3H-PC, which was calculated, for each sample, as follows:
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Values for the percentage secretion were arcsin-transformed and between-group differences in %PC secretion between warm-active and torpid groups, the presence or absence of agonist or the two incubation temperatures, were analysed using unpaired Student's t-tests (two-sample for means, assuming equal variance, P<0.05). For within-group comparisons (i.e. to determine if the agonist affected cells from one particular animal), the control and agonist-treated groups were from the same preparation of cells and incubated and treated in an identical manner and at the same temperature on the same plate. Therefore, the differences in PC secretion between control and agonist-treated wells were analyzed by paired t-tests (two-sample for means).
Q10 measurements were calculated for each time point at 37°C
and 4°C from the mean of the %PC secretion values obtained in each
experiment, using the following equation
(Schmidt-Nielson, 1997):
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cAMP production
The cAMP assay was adapted from McKinley and Hazel
(2000). Fresh cell isolates
were incubated overnight in DMEM + 10% FBS on bacteriological plates. After 22
h, the cells were centrifuged at 200 g and washed twice with
PBS. After the final spin, cells were resuspended in PBS containing 0.83 mg
ml-1 theophylline at 22°C and counted using a haemocytometer.
The cell suspension was diluted to approximately 1x106 cells
ml-1. 300 µl of cell suspension were sampled into Eppendorf
tubes (3x105 cells per tube). 30 µl of sample medium was
added to the control tubes and 30 µl of 1 mmol l-1 isoproterenol
(prepared in PBS containing theophylline) was added to each Eppendorf tube.
The tubes were incubated at either 37°C or 4°C for 15 min. After 20
min, 60 µl of 6% (w/v) trichloroacetic acid (TCA) was added to each tube
and the tubes plunged immediately into liquid nitrogen to stop the reaction.
The samples were thawed and neutralised with 200 µl of 2 mol l-1
KHCO3. The samples were centrifuged for 2 min at 17 000
g in an Eppendorf centrifuge and the supernatant collected.
The samples were then stored at -80°C for a maximum of 21 days. cAMP was
measured using a direct cAMP ELISA kit (Amersham Pharmacia Biotech, Canada).
cAMP production (pmole cell-1) was calculated from the data. Data
were analysed using paired and unpaired t-tests assuming equal
variance. Statistical significance was assumed at P<0.05.
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Results |
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Cell yield, purity and viability
When analysed using Trypan Blue, yields of
34.5±3.3x106 (mean ± S.E.M.,
N=15) and 29.3±3.3x106 (mean ±
S.E.M., N=10) viable alveolar type II cells
were obtained from each warm-active and torpid squirrel, respectively. Freshly
isolated warm-active squirrel cell suspensions were 96.6±0.4% (mean
± S.E.M., N=16) viable. Freshly isolated torpid
squirrel cell suspensions were 97.85±0.33% viable (N=12). Type
II cell suspensions from warm-active squirrels were 93.4±0.5% (mean
± S.E.M., N=10) viable after overnight incubations
at 37°C. Type II cell suspensions from torpid squirrels were
98.9±0.6% viable (mean ± S.E.M., N=10) after
overnight incubations at 4°C. As determined by the LDH assay, cell
viability remained above 90% after overnight incubations and for the duration
of all experiments, at both temperatures. Both the incubation temperature and
the presence or absence of agonists had no effect on cell viability, as
measured by the exclusion of the vital dye, Trypan Blue and the LDH
cytotoxicity assay.
Microscopy
An electron micrograph of the alveolar epithelium of the lung of a
golden-mantled ground squirrel Spermophilus lateralis is shown in
Fig. 1. The photograph shows an
alveolar type II cell, located in a crevice between alveoli, and demonstrates
the cuboidal shape, microvilli and presence of large osmiophilic lamellar
bodies in these cells.
|
Surfactant secretion
The effect of temperature on phosphatidylcholine secretion
Basal secretion after 0.5 and 1 h of incubation was significantly higher
when cells were incubated at a temperature matching the body temperature of
the squirrel from which they were isolated (37°C or 4°C), compared to
the alternative assay temperature (4°C or 37°C, respectively)
(Fig. 2). In type II cells
isolated from warm-active squirrels, basal secretion was 1.6- and 2.2-fold
higher at 0.5 and 1 h, respectively, at an assay temperature of 37°C
compared to 4°C. In type II cells isolated from torpid squirrels, basal
secretion was 1.8- and 1.7-fold higher at 0.5 and 1 h, respectively, at an
assay temperature of 4°C compared to 37°C. Consequently,
agonist-stimulated secretion was also significantly higher at an assay
temperature of 4°C than at 37°C in torpid squirrel cells at 0.5 and 1
h time points. The Q10 values obtained for the process of
surfactant secretion in type II cells isolated from warm-active and torpid
squirrels are shown in Table 1.
For cells from both warm-active and torpid squirrels, the presence of agonists
had no effect on the Q10 value of surfactant secretion. Cells from
torpid squirrels had a significantly lower Q10 value than that
observed in cells from warm-active squirrels (1.23 vs 0.86); however,
both Q10 values fell well below a Q10 of 2, which
suggests that surfactant secretion in squirrel type II cells is insensitive to
temperature.
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|
Temperature and the control of phosphatidylcholine secretion by
neurochemicals
In type II cells isolated from warm-active squirrels, the adrenergic
agonist, isoproterenol, significantly increased surfactant secretion above
basal levels after 1 h at 37°C, but not after 0.5 h at 37°C
(Fig. 2). When warm-active
squirrel type II cells were incubated at 4°C, isoproterenol significantly
increased surfactant secretion above basal levels after 1 h. Torpid squirrel
type II cells did not respond to the adrenergic agonist, isoproterenol, after
0.5 or 1 h at an assay temperature of 37°C; however, isoproterenol did
significantly increase surfactant secretion after 1 h at an assay temperature
of 4°C.
The cholinergic agonist, carbamylcholine chloride, did not significantly increase surfactant secretion above basal levels in type II cells isolated from warm-active ground squirrels, at assay temperatures of either 37°C or 4°C (Fig. 2). Moreover, type II cells isolated from torpid ground squirrels did not respond to carbamylcholine chloride when incubated at 37°C; however, carbamylcholine chloride did significantly increase surfactant secretion above basal levels in torpid squirrel cells when the cells were incubated at 4°C for 30 min.
cAMP production
Basal cAMP production was unaffected by either the state of the squirrel
from which the cells were isolated, or by the temperature at which the assay
was performed (Fig. 3).
Isoproterenol significantly increased cAMP production in type II cells
isolated from both warm-active and torpid squirrels at both 37°C and
4°C assay temperatures. When assayed at the body temperature of the
squirrel from which the cells were isolated, isoproterenol-stimulated
secretion was significantly higher in type II cells isolated from torpid
ground squirrels compared to warm-active squirrels.
|
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Discussion |
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PC secretion from type II cells isolated from torpid squirrels incubated at
4°C is almost double that measured from type II cells isolated from
warm-active squirrels incubated at 37°C. Therefore, despite a dramatic
drop in body temperature during torpor, the release of surfactant from type II
cells in torpid squirrels is maintained and even upregulated to levels above
those of type II cells from warm-active squirrels. Such an observation
suggests that squirrel type II cells and, particularly, their pathways of
surfactant secretion, are highly adapted to cope with temperature
fluctuations. Furthermore, the upregulation of surfactant secretion during
torpor highlights the importance of surfactant function to the lung even when
ventilatory rate and tidal volume are markedly reduced. The Q10
values obtained from type II cells isolated from both warm-active and torpid
squirrels and incubated at both 37°C and 4°C are given in
Table 1. In all experimental
groups, the Q10 values were significantly below 2, which confirms
that the process of surfactant secretion in squirrel type II cells is highly
insensitive to changes in incubation temperature. We reported a similar
observation (Q10=1.2) in type II cells isolated from warm-active
dunnarts and incubated at 37°C and 18°C
(Ormond et al., 2001). In
addition, the Q10 value of 0.85 obtained for type II cells isolated
from torpid squirrels is significantly lower than the value of 1.2 obtained
for type II cells isolated from warm-active squirrels. This may indicate that
during torpor, additional modifications occur at the cellular level to promote
the secretion of surfactant from squirrel type II cells. Such modifications
may include thermal acclimation of plasma membranes
(Hazel and Zerba, 1986
),
thermal modification of proteins or enzymes
(Storey, 1997
) or the
upregulation of receptors and signalling molecules
(Van Breukelen and Martin,
2002
) involved in the surfactant secretory and regulatory pathways
within the cell.
Although the process of surfactant secretion from squirrel type II cells is
relatively insensitive to temperature, short-term changes in assay temperature
significantly decreased surfactant secretion. PC secretion from type II cells
isolated from warm-active squirrels was significantly higher at an assay
temperature of 37°C than at 4°C. Conversely, PC secretion from type II
cells isolated from torpid squirrels was significantly higher at an assay
temperature of 4°C than at 37°C. Hence, basal surfactant secretion was
significantly higher when type II cells were incubated at a temperature
similar to the body temperature of the squirrel from which they were isolated,
whether 37°C or 4°C. The decreases in surfactant secretion from
warm-active squirrel type II cells incubated at 4°C compared to 37°C
could be attributed to a decrease in cellular metabolic rate at 4°C. A
similar decrease in PC secretion was observed in warm-active dunnart type II
cells incubated at 18°C compared to 37°C
(Ormond et al., 2001). We
observed a significant increase in PC secretion in type II cells isolated from
torpid squirrels and incubated at 4°C, however, and this suggests that
squirrel type II cells undergo a process of thermal acclimation, in
preparation for or during torpor, which results in the upregulation of
surfactant secretion. Therefore, the upregulation of surfactant secretion from
squirrel type II cells appears to be an adaptive characteristic of torpor.
Control of phosphatidylcholine secretion by an adrenergic
agonist
Warm-active squirrel type II cells appear to respond to isoproterenol at
both warm and cold incubation temperatures. This is similar to observations we
have previously made in type II cells isolated from bearded dragons and
warm-active dunnarts incubated at 18°C and 37°C
(Wood et al., 1999). In the
present study, the stimulatory response of squirrel type II cells to
isoproterenol after 1 h at 37°C (270% after 1 h) is similar to that
observed in rat type II cells at 37°C (300% after 1.5 h)
(Dobbs and Mason, 1979
). In
type II cells isolated from torpid squirrels, the response to isoproterenol is
small (125% after 1 h at 4°C) compared to that observed in warm-active
squirrel cells (270% after 1 h at 37°C). Although the relative roles of
the sympathetic and parasympathetic nervous systems during torpor are not yet
clear, it is generally accepted that during the deep torpor of hibernators the
activity of the sympathetic nervous system is dramatically reduced, if not
eliminated (Milsom et al.,
1999
). Hence, adrenergic agonists may not be an important
regulatory mechanism of surfactant secretion during deep torpor. Consequently,
squirrel type II cells may downregulate their response to adrenergic
stimulation by decreasing receptor number or the activity of enzymes in the
ß-adrenergic stimulatory pathway. Decreasing receptor number, however,
could potentially impair the initiation of arousal, which is accompanied by a
large increase in sympathetic activity
(Milsom et al., 1999
). Hence,
in order to enable a rapid response to the return of sympathetic activation,
it seems more likely that receptor number on alveolar type II cells is
maintained during torpor. Furthermore, we observed an increase in the
production of cAMP in response to isoproterenol in torpid squirrel type II
cells (Fig. 3), which indicates
that isoproterenol is binding to at least some ß-adrenergic receptors in
the plasma membranes of torpid cells. A decrease in cellular metabolic rate
and/or the activity of enzymes involved in ß-adrenergic receptor
signalling could also account for the smaller response to adrenergic
stimulation we observed in torpid squirrel type II cells; however, we have
observed that torpid squirrel type II cells have significantly higher levels
of basal PC secretion than warm-active squirrel type II cells, when assayed at
the body temperature of the animal from which the cells were isolated.
Therefore, the upregulation of basal secretion may be an adaptive response to
the decrease in adrenergic stimulation, and hence, agonist-stimulated
surfactant secretion, during torpor. Alternatively, the decrease in the
response of torpid type II cells to adrenergic stimulation may be due, at
least in part, to the upregulation of basal secretion during torpor, and
therefore a lower reserve capacity to respond to adrenergic agonists.
Control of phosphatidylcholine secretion by a cholinergic
agonist
Carbamylcholine chloride did not appear to stimulate surfactant secretion
very effectively in the squirrel, and this finding is consistent with
observations made in homeothermic mammalian type II cells. While
carbamylcholine chloride can stimulate surfactant secretion in vivo,
it does not appear to act directly on type II cells isolated from homeothermic
mammals (Dobbs and Mason,
1979). However, a significant increase in response to
carbamylcholine chloride was observed in torpid squirrels at an assay
temperature of 4°C after 1 h and this finding is consistent with
observations made in type II cells isolated from heterothermic mammals and
ectothermic animals. Type II cells isolated from dunnarts S.
crassicaudata, bearded dragons P. vitticeps, frogs Rana
catesbeiana and Australian lungfish Neoceratodus forsteri
respond directly to cholinergic agonists (Wood et al.,
1999
,
2000
). Furthermore, in
isolated bearded dragon type II cells, carbamylcholine chloride only
stimulated cells that were incubated at cold assay temperatures (18°C) and
not at warm assay temperatures (37°C)
(Wood et al., 1999
).
Similarly, in the present study, carbamylcholine chloride only stimulated
surfactant secretion in torpid squirrel cells incubated at 4°C, and not at
37°C. Furthermore, warm-active squirrel type II cells did not respond to
carbamylcholine chloride at either incubation temperature. Hence, the response
to carbamylcholine chloride appears to be highly temperature sensitive in
squirrel and lizard type II cells and appears to be only operational at cold
temperatures. The switch in the response of type II cells to cholinergic
factors at low incubation temperatures may be due to temperature-sensitive
cholinergic receptors or enzymes in the cholinergic signalling pathway.
Alternatively, low temperatures may reduce the activity of enzymes, such as
acetylcholinesterase, which break down acetylcholine and, therefore, may lead
to a relative increase in the amount of acetylcholine interacting with the
cholinergic receptors on type II cells
(Wood et al., 1999
).
In this study, type II cells isolated from warm-active squirrels did not
respond to cholinergic stimulation, which suggests that parasympathetic
control of surfactant secretion, through interactions with muscarinic
receptors on type II cells, is not crucial in warm-active animals. However, it
should be noted that parasympathetic control of surfactant secretion from type
II cells may still occur indirectly in warm-active animals in vivo.
Wood et al. (1997) postulated
that the parasympathetic nervous system can also stimulate surfactant
secretion in vivo via the stimulation of receptors on pulmonary
smooth muscle and the subsequent distortion of type II cells (mechanical
stimulation). In ground squirrels, the entrance into torpor is controlled by
the parasympathetic nervous system, which regulates the initial change in
heart rate that occurs before body temperature falls
(Milsom et al., 1999
). The
failure of warm-active squirrel type II cells to respond to cholinergic
agonists, at both warm and cold assay temperatures, suggests that
parasympathetic control may not be important in controlling surfactant
secretion during entry into torpor. Indeed, as body temperature begins to
fall, parasympathetic tone appears to be progressively withdrawn
(Milsom et al., 1999
). Some
authors believe that during deep torpor, parasympathetic influence is at a
minimum (Lyman and O'Brien,
1963
) or completely absent
(Milsom et al., 1993
). There
is also evidence, however, to suggest that both the sympathetic and
parasympathetic nervous systems are reduced in proportion to body temperature
(Q10=3) (Milsom et al.,
1993
). Furthermore, although the activity of the vagus nerve is
low during torpor, other studies support the conclusion that the
parasympathetic system still plays some role in cardiovascular control
(Milsom et al., 1999
). In this
study, torpor increased the sensitivity of squirrel type II cells to
cholinergic agonists, and this supports observations that some parasympathetic
tone is retained during deep torpor. Increasing the sensitivity of type II
cells to direct cholinergic stimulation during torpor may enable some
regulation of surfactant secretion, despite a reduced autonomic output.
cAMP production
We observed no differences in basal cAMP production between type II cells
isolated from torpid squirrels and warm-active squirrels assayed at 4°C
and 37°C, respectively. Hence, ground squirrels are still able to maintain
cAMP levels during torpor at 4°C. Furthermore, an acute temperature change
for the assay period (from 37°C to 4°C in warm-active squirrel cells
or from 4°C to 37°C in torpid squirrel cells) had no effect on cAMP
production. This is probably due to the short incubation time (15 min) of the
cAMP assay which, under culture conditions, may not have been long enough to
enable the type II cells to register the temperature change. Isoproterenol
significantly increases cAMP levels in type II cells isolated from warm-active
squirrels and torpid squirrels at both assay temperatures; however,
isoproterenol-stimulated cAMP production is significantly higher in type II
cells isolated from torpid ground squirrels compared to cells isolated from
warm-active squirrels when assayed at the temperature that matched the body
temperature of the squirrels from which the cells were isolated. This
indicates that the squirrel type II cells may be upregulating their response
to isoproterenol during torpor without increasing basal cAMP levels.
Furthermore, isoproterenol-induced cAMP production does not appear to
correspond directly to isoproterenol-stimulated PC secretion. cAMP is an
important indicator of ß-adrenergic receptor activity; however, there is
no evidence to suggest that increases in cAMP are directly linked to increases
in surfactant secretion. In fact, this study suggests that cAMP is not
directly involved in the stimulation of PC secretion, and the activities of
other signalling molecules may play a role here.
Conclusions
In this study, we observed that alveolar type II cells isolated from torpid
squirrels demonstrate an increased basal secretion of pulmonary surfactant.
These findings are supported by previous observations that surfactant PL
increases during cold-acclimation in ground squirrels
(Melling and Keough, 1981).
The process of surfactant secretion from squirrel type II cells is highly
resistant to temperature changes, as demonstrated by Q10 values of
0.85-1.2. Furthermore, when assayed at the temperature matching the body
temperature of the animal from which they were isolated, type II cells from
torpid squirrels demonstrated a higher basal surfactant secretion than those
isolated from warm-active squirrels. Therefore, the upregulation of surfactant
secretion from squirrel type II cells appears to be an adaptive characteristic
of torpor, as does the response of type II cells to cholinergic stimulation.
Increasing the sensitivity of type II cells to cholinergic (parasympathetic)
stimulation during torpor may enable surfactant secretion to be regulated or
enhanced, even when autonomic output is low. The response of squirrel type II
cells to cholinergic stimulation during torpor, but not euthermia, supports
observations that although parasympathetic tone may be significantly reduced
during deep torpor, it is still important in regulating some physiological
processes.
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References |
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