Ablation of the SERCA3 gene alters epithelium-dependent
relaxation in mouse tracheal smooth muscle
James
Kao1,
Christopher N.
Fortner1,
Lynne H.
Liu2,
Gary E.
Shull2, and
Richard J.
Paul1
Departments of 1 Molecular and Cellular
Physiology and 2 Molecular Genetics,
Biochemistry, and Microbiology, University of Cincinnati College of
Medicine, University of Cincinnati, Cincinnati, Ohio 45267-0576
 |
ABSTRACT |
Sarcoplasmic/endoplasmic reticulum
Ca2+-ATPase 3 (SERCA3), an isoform of the intracellular
Ca2+ pump that has been shown to
mediate endothelium-dependent relaxation of vascular smooth muscle, is
also expressed in tracheal epithelium. To determine its possible role
in regulation of airway mechanical function, we compared tracheal
contractility in gene-targeted mice deficient in SERCA3
(SERCA3
) with that in
wild-type tracheae. Cumulative addition of ACh elicited
concentration-dependent increases in isometric force (ED50 = 2 µM, maximum force = 8 mN/mm2) that were identical in
SERCA3
and wild-type
tracheae. After ACh stimulation, substance P (SP) elicited a transient
relaxation (42.6 ± 3.2%, n = 28)
in both tracheae. However, the rate of relaxation was significantly
(P < 0.04, n = 9) more rapid in the wild-type
[half-time (t1/2) = 34.3 s] than in the SERCA3
(t1/2 = 61.6 s)
trachea. The SP relaxation was reduced by rubbing the trachea,
indicative of epithelial cell involvement. This was verified using a
perfused trachea preparation. SP in the outside medium had no effect,
whereas SP in the perfusate bathing the epithelial side elicited a
relaxation. Nitric oxide synthase inhibition (0.2 mM
N
-nitro-L-arginine) reduced the
SP relaxation by 36.5 ± 12.5%, whereas the SP effect was abolished
by eicosanoid inhibition (10 µM indomethacin). ATP also elicited an
epithelium-dependent relaxation similar to SP but with a more rapid
relaxation in the SERCA3
trachea than in the wild-type trachea. Our results indicate that SERCA3
gene ablation does not directly affect smooth muscle, which is
consistent with the distribution of the isoform, but suggest that
SERCA3 plays a role in epithelial cell modulation of airway smooth
muscle function.
sarcoplasmic/endoplasmic reticulum calcium
ion-adenosinetriphosphatase 3; calcium ion-adenosinetriphosphatase; endoplasmic reticulum; substance P; adenosine triphosphate
 |
INTRODUCTION |
CA2+ plays a central role
in activation of both smooth muscle contractility (7) and
endothelium-dependent relaxation, for example via nitric oxide synthase
(5). Although the exact role of epithelial factors, such as eicosanoids
or nitric oxide, in modulating airway contractility is controversial
(13), intracellular Ca2+ is likely
a major factor in regulation of epithelial barrier and secretory
function. Thus the mechanisms underlying
Ca2+ homeostasis in these tissues
are central to regulation of airway function. The loading, storage, and
release of Ca2+ by the
sarcoplasmic/endoplasmic reticulum are determined in part by its
associated Ca2+-ATPases (12).
These Ca2+ pumps are known to
exist in several isoforms; in particular, the sarcoplasmic/endoplasmic
reticulum Ca2+-ATPase 3 (SERCA3)
isoform is known to be expressed in aortic endothelium and tracheal
epithelial cells (2, 3). Interestingly, SERCA3 is reported not to be in
tracheal smooth muscle (1). The role of this
Ca2+ pump isoform is not known
with certainty, particularly in view of the fact that SERCA2b is also
known to be expressed in these epi- and endothelial tissues and has
different Ca2+ and pH dependencies
(10). To better understand the role of SERCA3, we used a recently
developed mouse model in which the SERCA3 gene was ablated (9). In this
mouse, we showed that endothelium-dependent relaxation in aorta was
defective. Our hypothesis here is that the airway smooth muscle from
the SERCA3-deficient (SERCA3
) animals will be
unaffected, whereas epithelial-mediated responses will be altered
compared with those of wild-type tissues. To test this hypothesis, we
characterized the contractility of mouse tracheal smooth muscle and
epithelium-dependent relaxation of the trachea. In addition to the
first direct evidence for epithelial modulation of mouse airway smooth
muscle contractility, we show that SERCA3 can play a significant role
in modulating airways epithelial interaction.
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MATERIALS AND METHODS |
Tracheal tissue preparation.
Heterozygous mice were mated, and the pups were reared for 8-12
wk. Age-matched wild-type and SERCA3
pairs were then
selected for experimentation. Each mouse was killed by
CO2 asphyxiation. The trachea was
then quickly dissected and subsequently placed in physiological salt
solution (PSS) of the following composition (in mM): 118 NaCl, 4.73 KCl, 1.2 MgCl2, 0.026 EDTA, 1.2 KH2PO4,
2.5 CaCl2, and 11 glucose,
buffered with 25 NaH2CO3
to attain a pH of 7.4 at 37°C when bubbled with a mixture of 95%
O2 and 5%
CO2. Additional fat and tissue
were carefully removed from the trachea using spring scissors under a
light microscope. In some experiments, the epithelium was removed by
passing a silk thread or dental floss (Ultra Floss; Oral B, Redwood
City, CA) through the lumen of the trachea. Details of this procedure
and analysis of its effects on epithelium-dependent function are given in RESULTS.
Force measurements in tracheal ring
preparations. Tracheal rings were mounted isometrically
to force transducers (Harvard Apparatus, Hilliston, MA) using
0.012-in.-diameter wires such that force in the circumferential
direction was measured. The wire-mounted tracheae were then immersed in
a 50-ml organ bath filled with PSS and maintained at a constant
temperature of 37°C. Isometric force signals were acquired using a
data-acquisition program (AcqKnowledge, BioPac version 3.2). After
30-45 min of equilibration, two to three contraction/relaxation
cycles to ACh (10 µM) were performed until reproducible forces were achieved.
Perfused trachea preparations.
Tracheae were excised and dissected free of surrounding tissues as in
Tracheal tissue preparation. Each
trachea was tied with the ends around two cannulas made of 18-gauge
stainless steel tubing using 5-0 silk. The span of exposed trachea
between the cannulas was 5 mm. Differential pressure (
P) was
measured using two pressure transducers (Cobe) attached to wall taps
near the cannula openings and was recorded using the BioPac data-
acquisition system. Tracheae were placed in an open chamber, surrounded
by 10 ml of PSS. A separate chamber of 14 ml PSS was perfused and
recirculated at 16 ml/min through the lumen of the trachea using a gear
pump (Micropump). At this flow rate, baseline
P ranged from 1.1 to
1.5 mmHg for tracheae without stimulation.
Data analysis. Data are expressed as
means ± SE. The n value indicates
the number of mice used. For ring preparations, tracheae from wild-type
and gene-targeted mice were mounted in the same organ bath. Standard
ANOVA was used, with P < 0.05 taken
as evidence of statistical significance.
 |
RESULTS |
Tracheae were obtained from 10- to 12-wk-old mice that weighed ~32 g;
there were no differences in weight between mouse types, similar to
that previously reported (9). For comparison between gene-targeted and
wild-type animals, sibling pairs were used. The average values of the
gross morphological properties of the SERCA3
and wild-type
tracheae are presented in Table 1. In terms
of force-generating capacity, there were no differences in
cross-sectional area, which generally is linearly related to cellular
area and force capacity. There were no significant differences in
physical properties of these tracheae, which is consistent with that
previously reported for aortas from these mice (9).
Force-length behavior. To define the
optimal length for maximum active force, we measured the force-length
characteristics of the tracheal ring preparations. We recorded the
passive force (resting tension) and active force (10 µM ACh) and
determined the relationships from 14 tracheae, averaging eight length
points each. Figure 1,
A and
B, shows these relationships developed
from 111 experimental points. The resting tension of the trachea varied exponentially as
C/Cs
(Fig. 1A), where C is circumference, and Cs is the circumference of the trachea under
unloaded conditions. In Fig. 1B, the
active force (total force
passive force) is shown along with
its SE. The force-length relationship was very broad. We chose 1.4 × C/Cs
as our standard length, choosing a slightly suboptimal length to
improve resolution by reducing passive force relative to the active
force generated. At this length, maximum active forces per
cross-sectional area averaged 8 mN/mm2 and did not differ between
trachea types (Table 1).


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Fig. 1.
Force-length relationships of murine tracheal smooth muscle.
A: isometric force-length
relationships for wild-type mice tracheae stimulated with 10 µM ACh.
Top curve represents the total force
of the wire-mounted tracheae. Center
curve represents the tracheal resting tension, i.e., the passive force.
Bottom curve represents the active
ACh-induced force (total passive force) at different muscle
lengths. B: active isometric
force-length relationships shown in greater resolution (solid line) and
the boundaries (dotted lines) that represent the SE of the fit. In both
graphs, length was normalized to C/Cs [the
current circumference of trachea (C) compared with the
circumference under 0 load
(Cs)].
Data are from 14 tracheae and ~8 measurements at different lengths
per trachea.
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Concentration-active force
relationships. We chose ACh for our standard
contractile agonist because KCl, often used for reference contractions,
produced only a small contraction in the organ bath. Figure
2 shows a recording of a typical cumulative
concentration-force measurement. Thresholds were recorded at ~0.1
µM, and maximum forces occurred at 10-30 µM. Figure
3 graphically summarizes the data from
tracheae from five wild-type and
SERCA3
mice, which yielded
an average concentration-force relationship with an
ED50 of 2 µM. There were no
statistically significant differences in these relationships between
SERCA3-deficient and wild-type tracheae.

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Fig. 2.
Typical record of an experiment measuring cumulative ACh-active force
relationships for mouse tracheae. Arrows indicate the point in time at
which the concentration of ACh was increased (0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, and 30 µM, respectively). Horizontal scale arrow
represents 20 min; vertical scale arrow represents 10 mN.
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Fig. 3.
ACh concentration-active force of murine tracheal smooth muscle. Values
are averages ± SE (bars) for 5 tracheae from wild-type (WT; dotted
line) and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase
3-deficient [(SERCA3 ); knockout (KO); solid
line] mice. There were no statistically significant
differences.
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Characterization of epithelial airway smooth muscle
interactions. We tested substance P (SP), ATP, and
thrombin, typical endothelium-dependent vasodilators (4), for potential
epithelium-dependent effects. In the organ bath, when tracheae were
contracted with 10 µM ACh, SP and ATP each elicited a transient
relaxation, whereas thrombin had little effect. SP (0.01 µM) and ATP
(10 µM) were chosen as standards for our experiments because these
concentrations maximized the peak relaxation. Due to tachyphylaxis, we
did not attempt concentration-relaxation relationships. To assess
whether these responses were dependent on an intact epithelium, we
first tried to remove the epithelium using techniques similar to those
reported for removal of vascular endothelium. We tried several
techniques and were most successful with passing a thread (6-0
silk) through the lumen. Considerable care was required in this
protocol in that both the magnitude of the force and relaxation were
decreased by mechanical epithelial denudation. We were able to develop
a protocol used on 11 tracheae in which force after passage of the silk
thread was not significantly decreased (89 ± 6% of the force before denudation). In these tracheae, the peak relaxation to SP (0.01 µM) was decreased by 50 ± 4%
(n = 11) compared with that before our epithelium denudation protocol. In parallel time-course studies, the controls were decreased by 12 ± 3%,
significantly less than the silk thread-treated preparations
(n = 5, P = 0.026). We examined the histology from one pair of tracheae in which
the relaxation to SP was measured. After a control contraction and relaxation to SP, we removed the epithelium from one trachea and repeated the protocol. The tracheae were subsequently fixed in paraformaldehyde. Using three separate hematoxylin and eosin-stained sections of each trachea, epithelial cells were counted based on the
number of nuclei in the epithelial layer. The control trachea retained
100% of its original relaxation response to SP, and the epithelial
layer consisted of 567 ± 19 continuously adjacent cells in a cross
section of the tracheal ring. The silk thread-treated trachea retained
only 33% of its original SP response, and the epithelial layer
contained 135 ± 8 cells evenly distributed around the ring, with no
more than eight cells adjacent to each other. These results suggested
that at least part of the relaxation to SP was dependent on an intact epithelium.
To further investigate whether the SP relaxation was epithelium
dependent, we developed an apparatus for perfusing an isolated mouse
trachea and for measuring the pressure drop due to tracheal resistance.
This resistance is related to tracheal diameter and hence is a measure
of tracheal contractility. This method is similar to one developed by
Munakata et al. (11) for use in guinea pig trachea but was modified to
accommodate the smaller dimensions of mouse airways. In this
experiment, the trachea could be both perfused and superfused such that
only the luminal side or exterior was exposed to the test solution.
Figure 4A
shows a typical experiment. After application of ACh (10 µM) to the
exterior solution, a steady-state contraction was measured, as
indicated by the increased pressure drop across the tracheal
resistance. Application of SP to the exterior had no effect for at
least 30 min (n = 5). In contrast, addition of SP to the perfusion of the luminal side of the trachea elicited a relaxation similar in magnitude and time course to that
observed in the ring preparation. ATP (10 µM) applied to the external
bath (Fig. 4B) produced only a small
relaxation (26 ± 9%) compared with luminal administration
(76 ± 3%; n = 4).


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Fig. 4.
Response of murine trachea to perfused or superfused substance P (SP)
or ATP. A: typical record showing that
SP-induced relaxation only occurs with luminal perfusion.
B: typical record showing ATP-induced
relaxation is significantly greater with luminal perfusion than
superfusion. C, contraction with 10 µM ACh; W, washout. Subscripts
denote extraluminal (e) or intraluminal (i) administration of agents.
Agents were added at the points indicated by the arrows and were not
removed until the points indicated by the "washout" arrows.
Horizontal scale arrow represents 20 min; vertical scale arrow
represents 0.5 mmHg differential pressure.
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To determine whether the differences in responses between luminal
perfusion and superfusion were related to epithelial barrier or
secretory function, we developed methods to remove the epithelium while
the trachea remained mounted in the perfusion apparatus. To remove the
epithelium from the trachea while mounted in our perfusion system, we
passed a length of dental floss (Oral B Ultra Floss) through the lumen
of the trachea. For three tracheae, this treatment reduced the
relaxation to SP from 60 ± 6% of the steady-state contraction to
15 ± 2%. These experiments provide additional evidence for an
epithelial dependence of the responses to SP and ATP.
Characterization of the SP relaxation.
Pretreatment of the ring preparation for 20 min with the nitric oxide
synthase inhibitor N
-nitro-L-arginine
(0.2 mM) decreased the relaxation induced by SP in ACh-contracted
trachea by 36 ± 12% (n = 9).
Pretreatment with 10 µM indomethacin increased the force of ACh
contraction similarly, by 36 ± 8% in wild-type and by 38 ± 10% in knockout tracheae. Importantly, indomethacin abolished the
SP-induced relaxation in all tracheae
(n = 5 wild type; n = 5 SERCA3
) studied. Because the extent of relaxation
is often inversely related to the magnitude of contraction, it is
possible that part of the decrease with indomethacin might be
attributable to this factor. As a control, we studied SP relaxation
using 30 mM ACh, a concentration at which force was 150% greater than
our reference contraction (10 µM ACh) used for the measurements of SP
effects. We found that the SP relaxation, expressed as a percent of the developed force, was independent of ACh concentration or force generated in either wild-type or
SERCA3
tracheae
(n = 3 each). In absolute terms, the magnitude of
the peak relaxation was actually greater at 30 mM ACh, but,
importantly, there were no differences between wild-type and
SERCA3
tracheae. Thus the
complete inhibition by indomethacin would suggest that the eicosanoid
pathway is the major pathway in the epithelium-dependent, SP-induced relaxation.
Comparison of wild-type and SERCA3
tracheae.
Figure 5 shows typical SP responses in
SERCA3
tracheae, and
averaged properties for 9-14 pairs are presented in Table
2. After ACh stimulation, SP elicited a
transient relaxation that was similar in magnitude for tracheae from
wild-type (42.0%, n = 14) and
SERCA3
(43.1%,
n = 14) mice. However, the rate of
relaxation was significantly (P < 0.04, n = 9) more rapid in the
wild-type [half-time
(t1/2) = 34.3 s] than in the SERCA3
(t1/2 = 61.6 s)
tracheae. To ensure simultaneous addition of SP, wild-type and
SERCA3
tracheae were
mounted in the same bath. It is possible that some interaction between
these tissues could occur, but with the dilution associated with a
50-ml bath (a dilution factor >20,000), we feel this is unlikely. We
tested for such effects by studying wild-type and
SERCA3
tracheae in separate
organ baths. There was no difference in the relaxation to SP from that
observed when mounted in the same bath. This suggests that differences
in time course were not due to diffusion of a relaxing factor from one
trachea to the other. Moreover, when an epithelium-denuded and an
intact trachea were placed in the same bath, the intact trachea relaxed
normally to SP while the denuded trachea did not relax at all. Thus the
differences in time courses do not reflect effects of diffusion of an
epithelium-dependent factor.

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Fig. 5.
Time course of SP-induced relaxation of
SERCA3 (solid line) and
wild-type (dotted line) tracheae. Representative tracings for typical
SERCA3 and wild-type
tracheae precontracted with 10 µM ACh and subsequently treated with
0.01 µM SP are shown. Recordings were scaled such that the magnitudes
of the peak relaxations were identical. Note that the average forces
per cross-sectional area did not differ between mouse types. Horizontal
scale arrow represents 10 min; vertical scale arrow represents 5 mN.
Wild-type trachea achieves peak relaxation well before that of the
SERCA3-deficient (SERCA3 )
trachea.
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We also characterized the relaxation to ATP (10 µM). A typical
experimental record is shown in Fig. 6.
These data are summarized in Table 3.
Interestingly, the time courses between wild-type and knockout tracheae
were reversed from that observed for SP in that the knockout trachea
relaxed faster than the wild type. In five of the eight pairs studied,
the t1/2 was
nearly two times as great in the wild-type than
SERCA3
tracheae, whereas in
three cases, the
t1/2 values were
similar. This trend was not statistically significant due to
variability in the absolute value of the relaxation times (21-244
s).

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Fig. 6.
SP- and ATP-induced relaxation of murine tracheal smooth muscle.
Representative tracings for SERCA3 (solid line) and wild-type (dotted
line) tracheae precontracted with 10 µM ACh and treated with 0.01 µM SP and subsequently with 10 µM ATP are shown. Horizontal scale
arrow represents 10 min; vertical scale arrow represents 5 mN.
Time-to-peak relaxation in response to SP is shorter in the wild-type
trachea (as in Fig. 5), whereas, in response to ATP, it is greater than
that of SERCA3
trachea.
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 |
DISCUSSION |
There are few experimental data on mouse tracheal function, and our
first goal was to characterize its contractility. It is likely that,
with the generation of mouse transgenic and gene-ablated models, it
will become an important preparation for study of airway regulation.
Its force-length relationships are similar to other smooth muscles.
Active isometric force per cross-sectional area was also not unusual
and in the range reported for other mouse smooth muscles, e.g., aorta
(14). A second objective of this study was to determine if
epithelium-dependent modulation of contractility could be demonstrated.
Although vascular smooth muscle-endothelial cell interactions are well
characterized (4), there is less evidence for such phenomena in
airways. Our evidence, based on both mechanical denudation of tracheal
epithelium and side-specific delivery of agonists, indicates that the
SP and ATP relaxation of an ACh contracture are epithelium-dependent
responses. Although our data show a relaxation dependent on epithelium,
there is no general consensus on the nature of such dependency. The
current controversy centers on whether the epithelium "barrier"
or "secretory" function is the major factor (13). Our data
strongly support the latter, particularly in the case of SP.
Application of SP to the serosal side with more direct access to the
smooth muscle component of the trachea had no effect, whereas luminal
infusion elicited relaxation. These results would also argue against
ascribing these results to nonepithelial differences such as in the
differences in localization of degradative enzymes, such as peptidases,
in the case of SP, or for conversion of ATP to adenosine. Moreover, adenosine (up to 10 µM) had no effect on the ring preparations (n = 4). Thus the studies on the perfused trachea
preparations make any barrier or mechanism involving differential
enzyme localization unlikely to explain our results. In conjunction
with the studies on the ring preparations, our data strongly suggest
that the relaxation elicited by either SP or ATP is an
epithelium-dependent, secretory function.
Our third objective was to identify the role of SERCA3 in epithelial
function in the airway. SERCA3 belongs to the P-type superfamily of ion
transport ATPases. The tissue specificity and cell-type distribution of
SERCA3 is limited, in contrast to the near-universal distribution of
SERCA2b. It is reported (3, 15) to be present in endothelial cells, in
epithelial cells of the trachea, intestine, and salivary glands, and in
platelets, mast cells, and lymphocytes. SERCA3 has a lower
Ca2+ affinity and a higher pH
optimum than SERCA2b (10). This is likely of functional significance,
since SERCA2b is also found in all cells known to contain SERCA3. The
limited expression of SERCA3 suggests that it may be involved in
tissue-specific signaling. This is in contrast to the near-universal
distribution of SERCA2b, which suggests that it likely is involved with
a broad-based function in Ca2+
homeostasis essential for cell viability. Given the differences in
biochemical parameters between SERCA3 and SERCA2b, one might also
anticipate that they would operate in different cellular environments
or compartments. With respect to smooth muscle function, the ACh
concentration-force relationships for both wild-type tracheae and
SERCA3
tracheae were
similar. This is consistent with the reported distribution of SERCA3,
indicating that it is not found in tracheal smooth muscle (1).
Comparison of the SP- and ATP-induced relaxation wild-type and
SERCA3
tracheae suggests a
role for the SERCA3 pump in modulation of epithelium-dependent
processes. The major effect observed was on the rate of the relaxation.
For SP, the relaxation
t1/2 was
considerably shorter in the wild-type than that in the
SERCA3
trachea. The
relaxation was abolished by eicosanoid pathway inhibition and was
partially blocked by inhibition of nitric oxide synthase. Both the
eicosanoid and nitric oxide pathways, implicated by these studies, are
dependent on intracellular Ca2+.
Thus one possible mechanism to explain the slower rate of relaxation is
that, in the absence of SERCA3, there is less
Ca2+ available for release by the
endoplasmic reticulum.
In response to ATP, there was a trend to the opposite direction, with
the t1/2 being
the same or faster in the
SERCA3
trachea than in the
wild type. One might anticipate that differences in epithelial
processing of SP and ATP, for example conversion of ATP to adenosine
vs. SP mediator release, could underlie the differences in time
courses. Adenosine did not relax these preparations, so the effects of
ATP were likely mediated by purinergic receptors. A speculative but
intriguing basis for this difference lies in the possibility of a
differential localization of the SERCA isoforms. In tracheal epithelial
cells, it is not known whether the different SERCA isoforms serve the
same or different subcompartments of the endoplasmic reticulum.
However, in salivary epithelial cells, SERCA2b and SERCA3 do localize
to separate compartments (8). It is possible that the signaling pathway
for subcompartments associated with SERCA2b may be different from that
associated with SERCA3 and may underlie the differences in relaxation
between wild-type and
SERCA3
aortas to SP and ATP.
The physiological significance of an epithelium-dependent relaxation to
SP is also not clear, but our results are not unprecedented. In organ
bath studies, rubbing the epithelial layer with cotton wool blunted the
SP relaxation of rat trachea (6).
In conclusion, we have shown that mouse tracheal muscle shows an
epithelium-dependent relaxation involving an indomethacin-inhibitable factor. In tracheae from
SERCA3
mice, smooth muscle
contractility is unchanged, but the time course of the
epithelium-dependent relaxation to SP is altered. Our results are
consistent with earlier studies on endothelium-dependent relaxation in
SERCA3
aorta. These results
clearly implicate a unique role in endo(epi)thelial cell signal
transduction for the SERCA3 isoform.
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ACKNOWLEDGEMENTS |
This work was supported by an Undergraduate Internship of the
Howard Hughes Medical Institute; the Department of Biological Sciences,
University of Cincinnati (to J. Kao); MD/PhD Scholar Award (to C. N. Fortner); and National Institutes of Health Grants HL-61974 (to G. E. Shull and R. J. Paul), DK-50594 (to G. E. Shull), and HL-54829 (to R. J. Paul).
 |
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: R. J. Paul,
Dept. of Molecular and Cellular Physiology, Univ. of Cincinnati College
of Medicine, 231 Bethesda Ave., Cincinnati, OH 45267-0576 (E-mail:
richard.paul{at}uc.edu).
Received 16 October 1998; accepted in final form 30 March 1999.
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