Am J Physiol Lung Cell Mol Physiol 294: L787–L796, 2008.
First published February 1, 2008; doi:10.1152/ajplung.00461.2007.
Regulation of sarcoplasmic reticulum Ca2⫹ reuptake in porcine airway
smooth muscle
Venkatachalem Sathish,1 Figen Leblebici,2 Sertac N. Kip,1 Michael A. Thompson,2
Christina M. Pabelick,1,2 Y. S. Prakash,1,2 and Gary C. Sieck1,2
Departments of 1Physiology and Biomedical Engineering, and 2Anesthesiology, Mayo Clinic College of Medicine, Rochester,
Minnesota
Submitted 6 November 2007; accepted in final form 28 January 2008
sarco(endo)plasmic reticulum calcium-ATPase; phospholamban; calmodulin; calmodulin kinase
tant for replenishment of SR Ca2⫹ stores during agonist stimulation. SR Ca2⫹ reuptake occurs via the sarco(endo)plasmic
reticulum Ca2⫹-ATPase (SERCA). Accordingly, regulation of
SERCA controls the extent of SR Ca2⫹ replenishment and,
thus, subsequent [Ca2⫹]i response to agonist.
Studies in cardiac and slow-twitch skeletal muscles have
long established that SERCA can be regulated by phospholamban (PLN), such that phosphorylation of PLN (e.g., by cyclic
nucleotides) disinhibits SERCA (5, 9, 37). Although PLN
expression and phosphorylation under physiological conditions
have been demonstrated in gastrointestinal (27, 28) and vascular smooth muscles (6, 8, 18, 25, 30, 59), data on the role of
PLN in ASM are very limited (38).
Phosphorylation of PLN can occur via cAMP-dependent
protein kinase or Ca2⫹/calmodulin (CaM)-dependent protein
kinase (CaMKII) (57, 60). Previous studies have documented
the existence of CaM kinase and a 17-kDa substrate polypeptide, both of which are associated with SR vesicles from ASM
(10). Furthermore, a CaM-dependent kinase has been shown to
phosphorylate and, thereby, activate SERCA in porcine coronary artery smooth muscle (18). However, SERCA modulation
by CaMKII has not been reported in ASM. In the present study,
using porcine ASM cells, we examined the hypothesis that
PLN is present in ASM and regulated by CaMKII.
In previous studies, we demonstrated that agonists such as
ACh induce propagating [Ca2⫹]i oscillations, which represent
repetitive SR Ca2⫹ release and reuptake (via IP3 channels during
initiation and RyR channels during maintenance) (24, 45, 56).
Oscillation amplitude reflects the size of the local SR store (Ca2⫹
content) and oscillation frequency the time course for SR release/
reuptake (43, 56). We and others have shown that [Ca2⫹]i oscillations allow for sustained force responses with limited SR Ca2⫹
stores (2, 47, 56) at different agonist concentrations. Accordingly,
SR Ca2⫹ reuptake is important in regulating ASM contractility.
Given the potential role of CaM/CaM kinase in [Ca2⫹]i regulation, in this study, we examined whether ACh-induced [Ca2⫹]i
oscillations involve CaM and/or CaMKII.
IN AIRWAY SMOOTH MUSCLE (ASM), regulation of intracellular
Ca2⫹ concentration ([Ca2⫹]i) under basal conditions and with
agonist stimulation is important in controlling airway tone.
Sarcoplasmic reticulum (SR) Ca2⫹ release and reuptake is a
key component in ASM [Ca2⫹]i regulation and agonist response (22, 43, 52). In ASM, SR Ca2⫹ release involves inositol
trisphosphate (IP3) and ryanodine receptor (RyR) channels (1,
43, 45). Ca2⫹ influx, via multiple mechanisms (22), is impor-
Reagents for electrophoresis were obtained from Bio-Rad Laboratories (Hercules, CA), anti-PLN monoclonal antibody and anti-phosphorylated (Ser16) PLN polyclonal antibody from Upstate Biotechnology (Lake Placid, NY), anti-phosphorylated (Thr17) PLN and PLN
small interfering RNA (siRNA) from Santa Cruz Biotechnology
Address for reprint requests and other correspondence: G. C. Sieck, Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, Rochester, MN 55905 (e-mail: [email protected]).
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.
http://www.ajplung.org
MATERIALS AND METHODS
Chemicals
1040-0605/08 $8.00 Copyright © 2008 the American Physiological Society
L787
Downloaded from http://ajplung.physiology.org/ by 10.220.32.247 on June 18, 2017
Sathish V, Leblebici F, Kip SN, Thompson MA, Pabelick CM,
Prakash YS, Sieck GC. Regulation of sarcoplasmic reticulum Ca2⫹
reuptake in porcine airway smooth muscle. Am J Physiol Lung Cell
Mol Physiol 294: L787–L796, 2008. First published February 1, 2008;
doi:10.1152/ajplung.00461.2007.—Regulation of intracellular Ca2⫹
concentration ([Ca2⫹]i) in airway smooth muscle (ASM) during
agonist stimulation involves sarcoplasmic reticulum (SR) Ca2⫹ release and reuptake. The sarco(endo)plasmic reticulum Ca2⫹-ATPase
(SERCA) is key to replenishment of SR Ca2⫹ stores. We examined
regulation of SERCA in porcine ASM: our hypothesis was that the
regulatory protein phospholamban (PLN) and the calmodulin (CaM)CaM kinase (CaMKII) pathway (both of which are known to regulate
SERCA in cardiac muscle) play a role. In porcine ASM microsomes,
we examined the expression and extent of PLN phosphorylation after
pharmacological inhibition of CaM (with W-7) vs. CaMKII (with
KN-62/KN-93) and found that PLN is phosphorylated by CaMKII. In
parallel experiments using enzymatically dissociated single ASM
cells loaded with the Ca2⫹ indicator fluo 3 and imaged using fluorescence microscopy, we measured the effects of PLN small interfering RNA, W-7, and KN-62 on [Ca2⫹]i responses to ACh and direct
SR stimulation. PLN small interfering RNA slowed the rate of fall of
[Ca2⫹]i transients to 1 ␮M ACh, as did W-7 and KN-62. The two
inhibitors additionally slowed reuptake in the absence of PLN. In
other cells, preexposure to W-7 or KN-62 did not prevent initiation of
ACh-induced [Ca2⫹]i oscillations (which were previously shown to
result from repetitive SR Ca2⫹ release/reuptake). However, when
ACh-induced [Ca2⫹]i oscillations reached steady state, subsequent
exposure to W7 or KN-62 decreased oscillation frequency and amplitude and slowed the fall time of [Ca2⫹]i transients, suggesting
SERCA inhibition. Exposure to W-7 completely abolished ongoing
ACh-induced [Ca2⫹]i oscillations in some cells. Preexposure to W-7
or KN-62 did not affect caffeine-induced SR Ca2⫹ release, indicating
that ryanodine receptor channels were not directly inhibited. These
data indicate that, in porcine ASM, the CaM-CaMKII pathway regulates SR Ca2⫹ reuptake, potentially through altered PLN phosphorylation.
L788
CALMODULIN AND SMOOTH MUSCLE Ca2⫹
(Santa Cruz, CA), anti-SERCA2 ATPase monoclonal antibody from
Abcam (Cambridge, MA), DMEM-Ham’s F-12 medium, Pen-Strep,
Lipofectamine 2000, fura 2-AM, and fluo 3-AM from Invitrogen
(Carlsbad, CA), and KN-62, KN-93, and W-7 from Calbiochem (San
Diego, CA). All other chemicals were obtained from Sigma (St.
Louis, MO) unless otherwise noted.
Cell Preparation
PLN Knockdown by siRNA
A 20- to 25-nucleotide target-specific siRNA corresponding to
human PLN mRNA was selected for PLN knockdown. Porcine ASM
were isolated as described above and grown in growth medium
consisting of DMEM-Ham’s F-12 medium containing 10% fetal
bovine serum and 1% Pen-Strep until they reached ⬃70% confluence.
Cells were then rinsed in serum- and antibiotic-free DMEM-Ham’s
F-12 medium and transfected using 50 nM siRNA and Lipofectamine
2000. Fresh growth medium was added 6 h after addition of siRNA,
and the cells were analyzed 48 h after transfection. The efficacy of
siRNA knockdown was verified by Western analysis of decreased
PLN expression.
Preparation of SR Membrane Vesicles
SR membrane vesicles were isolated from ASM according to
previously described procedures (18, 53). Briefly, the smooth muscle
layer from fresh porcine trachea was minced well on ice and suspended in ice-cold 10 mM NaHCO3 buffer (pH 6.8) prepared in
Ca2⫹-free water, 20 ␮g/␮l leupeptin, and 100 ␮g/␮l trypsin inhibitor.
After three homogenization cycles for 15 s each with a Dounce
(Polytron) homogenizer, the homogenate was centrifuged for an
additional 10 min at 1,000 g (4°C) using a Beckmann JA-17 rotor. The
supernatant was collected and kept on ice, and the pellet was reconstituted with 4 vol of ice-cold NaHCO3 buffer and then recentrifuged.
The supernatant was decanted and combined with the first supernatant
and centrifuged for 20 min at 8,000 g (4°C). After addition of 0.6 M
KCl, the pooled supernatants were further subjected to high-speed
centrifugation (40,000 g for 1 h at 4°C) using a Beckmann Ti70 rotor.
The final supernatant was discarded, and the pellet was suspended in
a small volume of 10 mM Tris-maleate (pH 6.8) buffer containing 100
mM KCl and protease inhibitors. After determination of protein
content of this purified SR, samples were divided into aliquots, frozen
in liquid nitrogen, and stored at ⫺80°C.
Determination of Ca2⫹ Uptake
After determination of the specific activity of the 45Ca2⫹ isotope
(catalog no. NEZ013, Perkin Elmer), the loading medium (LM),
consisting of 50 mM Tris-maleate (pH 6.8), 5 mM MgCl2, 5 mM
NaN3, 120 mM KCl, 0.1 mM EGTA, 5 mM potassium oxalate, 0.025
mM ruthenium red, and 8 ⫻ 103 cpm/nmol 45Ca2⫹, was prepared. The
final concentration of KN-62, KN-93, and W-7 was 10 ␮M, with
controls containing vehicle alone, and the LM (with or without
KN-62, KN-93, or W-7) was then incubated for 3 min at 37°C. Before
the addition of SR microsomes, duplicate 200-␮l aliquots were drawn
AJP-Lung Cell Mol Physiol • VOL
Phosphorylation Studies
The phosphorylation assay medium (50 ␮l total volume) contained
50 mM HEPES (pH 7.4), 10 mM MgCl2, 0.1 mM CaCl2, 0.1 mM
EGTA, and SR (25 ␮g of protein). The final KN-62 and KN-93
concentration was 10 ␮M, with controls containing buffer alone in the
presence or absence of Ca2⫹ in the assay medium. The phosphorylation reaction was initiated by addition of 10 mM ATP after preincubation of the phosphorylation assay medium for 3 min at 37°C.
Reactions were terminated after 3 min by addition of 15 ␮l of SDS
sample buffer, and SR proteins were separated by SDS-PAGE using
a 15% Criterion Gel System (Bio-Rad Laboratories). Proteins were
transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories) for 60 min. The membranes were blocked for 1 h with 5%
milk in Tris-buffered saline containing 0.1% Tween, incubated with
appropriate primary antibody overnight at 4°C, washed with Trisbuffered saline containing 0.1% Tween, detected with horseradish
peroxidase-conjugated secondary antibodies, and developed with Supersignal West Pico Chemiluminescent Substrate (Pierce Chemical,
Rockford, IL).
[Ca2⫹]i Imaging
Freshly dissociated porcine ASM cells plated on collagen-coated
coverslips were incubated in 5 ␮M fura 2-AM for 45 min at room
temperature and then washed with HBSS (2 mM Ca2⫹). Coverslips
were placed in an open microscope imaging chamber (model RC25F,
Warner Instruments) and visualized with a real-time fluorescence
imaging system (MetaFluor, Universal Imaging, Downingtown, PA)
on a Nikon Diaphot inverted microscope (Fryer Instruments, Edina,
MN). The dye was alternately excited at 340 and 380 nm (Lambda
10-2 filter changer, Sutter Instrument, Novato, CA), and emissions
were collected with a 510-nm barrier filter. Images were acquired at
1.33 Hz with a Photometric Cascade digital camera system (Roper
Scientific, Tucson, AZ), and results are expressed as the ratio of
emissions at 340 nm to emissions at 380 nm. [Ca2⫹]i was quantified
from fura 2 levels with use of previously described calibration
procedures (19, 48). Cells were initially perfused with HBSS, and
baseline fluorescence was established. [Ca2⫹]i responses of 10 –15
cells per chamber were obtained using individual, software-defined
regions of interest.
In separate experiments requiring rapid acquisition of [Ca2⫹]i
transients, ASM cells on coverslips were loaded with 5 ␮M fluo 3-AM
(which, in contrast to fura 2, is nonratiometric) and imaged at 30 Hz
using real-time confocal microscopy (Noran Odyssey XL) as previously described (45). [Ca2⫹]i was quantified by mapping measured
fluorescence intensity to known Ca2⫹ levels with use of empirical
calibrations as described previously (45).
294 • APRIL 2008 •
www.ajplung.org
Downloaded from http://ajplung.physiology.org/ by 10.220.32.247 on June 18, 2017
The technique for isolation of porcine ASM cells has been previously described (24, 45, 56). Briefly, fresh porcine tracheas obtained
from an abattoir were transported in ice-cold Hanks’ buffered saline
solution (HBSS) containing 10 mM HEPES (pH 7.4; Life Technologies). The smooth muscle layer was excised and minced with scissors.
For biochemical work, these tissue fragments were processed as
described below. For single-cell work, tissue fragments were incubated with gentle agitation at 37°C with 20 U/ml papain and 2,000
U/ml DNase (Worthington Biochemical, Freehold, NJ). After 1 h, 0.8
mg/ml type IV collagenase (Worthington Biochemical) was added for
40 min. The dissociated cells were then triturated, centrifuged, and
plated on collagen-coated coverslips for imaging.
from treatment and control tubes for assessment of blank counts per
minute at time 0. The SR was prepared at a final concentration of 0.1
mg/ml in a phosphorylation medium containing 250 mM HEPES, 50
mM MgCl2, and 1 mM CaCl2, incubated for 2 min at 37°C, and then
combined with LM for further incubation at 37°C for 3 min. Immediately after the addition of 50 mM ATP, 45Ca2⫹ uptake into SR was
monitored over a 30-min period by transfer of the 200-␮l aliquots at
5, 15, 30 min onto GF/B 24-mm Whatmann filters. The filters were
vacuum-dried, and reactions were stopped by the addition of ice-cold
10 mM Tris-maleate (pH 6.8) wash buffer. Filters were then rinsed
five times with 5 ml of the wash buffer, air-dried, and immersed in 10
ml of Ultima-Gold scintillation liquid (Perkin Elmer) for measurement of counts per minute using a Beckmann scintillation counter.
Background correction was made by subtraction of values obtained
after incubation of microsomes in the absence of ATP for control
(without agonist) and inhibitor (with agonist). Values were calculated
as percent inhibition at each time point. The protein content of each
aliquot was then determined to further normalize the actual 45Ca2⫹
uptake in nanomoles per milligram of protein (18, 53, 67).
CALMODULIN AND SMOOTH MUSCLE Ca2⫹
L789
Experimental Protocols
RESULTS
Role of CaM and CaMKII in [Ca2⫹]i oscillations. The protocol for
establishing ACh-induced [Ca2⫹]i oscillations in ASM cells has been
previously published (45). We previously established that [Ca2⫹]i
oscillations represent repetitive SR Ca2⫹ release and reuptake and do
not require plasma membrane Ca2⫹ influx or efflux for initiation (45)
and that Ca2⫹ influx serves to replete SR Ca2⫹ stores. In this regard,
the effect of CaM on the plasma membrane Ca2⫹-ATPase is well
known. Therefore, to exclude any confounding effects of CaM or
CaMKII on plasma membrane Ca2⫹ regulatory mechanisms, we
conducted the following experiments in 0 Ca2⫹ HBSS containing 1
mM La3⫹. Cells were washed in HBSS without La3⫹ for ⱖ15 min.
ASM cells were exposed to 1 ␮M ACh (ED50 for oscillations), and
persistence of steady-state oscillations over a 2-min period was
confirmed. {We previously demonstrated that [Ca2⫹]i oscillations
persist for the duration of agonist stimulation (45).} During oscillations, 25 ␮M W-7 (to inhibit CaM) or 1 ␮M KN-62 (CaMKII
inhibitor) was added, and the response over the next 5 min was
observed. Finally, cells were reperfused with ACh only, and the
[Ca2⫹]i response was evaluated over the next 15 min. For determination of the effects of CaM and CaMKII antagonists on initiation of
ACh-induced [Ca2⫹]i oscillations, ASM cells in which [Ca2⫹]i oscillations had been verified were washed in HBSS for 15 min, incubated
in 0-Ca2⫹ HBSS (and La3⫹) containing 25 ␮M W-7 or 1 ␮M KN-62
for 5 min, and then exposed to 1 ␮M ACh in the continued presence
of either inhibitor. For control experiments, the initial ACh response
was followed by a 5-min rinse with 0-Ca2⫹ HBSS with La3⫹ followed
by a second treatment with ACh (in the absence of W-7 or KN-62).
For each experiment, peak and trough [Ca2⫹]i levels and the rate of
fall of individual oscillations (normalized to oscillation amplitude and
averaged over 5 oscillations) were measured. As previously demonstrated (45), the peak reflects net release of SR Ca2⫹ stores, and the
trough represents the point at which maximal SR Ca2⫹ reuptake had
occurred, before initiation of the next oscillation. Oscillation fall time
(normalized for amplitude and measured over a fixed time period of
100 ms from the peak of the [Ca2⫹]i transient) was indicative of
SERCA activity.
In ASM cells that did not display [Ca2⫹]i oscillations but, rather, a
transient increase in [Ca2⫹]i followed by a sustained (but lower)
plateau, the rate of fall of the [Ca2⫹]i response starting from the peak
value was estimated using a single-exponential fitting curve (Origin
statistical software).
Role of CaM and CaMKII in SR Ca2⫹ reuptake. The effect of W-7
or KN-62 on the [Ca2⫹]i response to 5 mM caffeine [ED50 for SR
Ca2⫹ release via RyR channels (23)] was examined in separate
experiments. ASM cells were bathed in 0-Ca2⫹ HBSS containing
La3⫹ and then exposed to 5 mM caffeine for 2 min. The caffeine
was washed out with HBSS for 15 min, and cells were exposed to
25 ␮M W-7 or 1 ␮M KN-62 (5 min) and then to 5 mM caffeine.
In control experiments, a second response to caffeine in the
absence of either inhibitor was verified. For each experiment, peak
and fall time (normalized for amplitude) of the [Ca2⫹]i response
were measured.
Effect of CaM and CaMKII Inhibition on SR Ca2⫹ Reuptake
In SR microsomes from porcine ASM, addition of ATP (to
activate SERCA) progressively increased 45Ca2⫹ uptake over
30 min (Fig. 1). Preincubation of microsomes in 10 ␮M W-7,
KN-62, or KN-93 significantly slowed and decreased the
extent of Ca2⫹ uptake compared with controls in the absence of
these inhibitors (P ⬍ 0.05; Fig. 1). Background subtraction for
nonspecific inhibitor effects in the absence of ATP was performed before comparison of inhibitor effects with controls in
the presence of ATP. There was no significant difference in the
inhibitory effects of W-7, KN-62, and KN-93 on Ca2⫹ uptake
in microsomes.
Western analysis using anti-PLN antibody revealed the presence of PLN in porcine SR microsomes (Fig. 2). At baseline
(i.e., no stimulation), PLN showed a moderate level of phosphorylation at Ser16 (which is believed to be modulated by
protein kinase A), but only minimal phosphorylation at Thr17
(the site targeted by CaMKII). Addition of ATP in the presence
of Ca2⫹ and (endogenous) CaM in vitro resulted in significantly greater phosphorylation at Thr17 (Fig. 2). Preincubation
with KN-93 or KN-62 significantly reduced Thr17 phosphorylation. There was no significant difference between the effects
of KN-93 and KN-62. In comparison, the extent of Ser16
phosphorylation was largely unaffected by KN-93 or KN-62.
Confirmatory analysis for SERCA protein revealed expression
of the SERCA2 protein (Fig. 2).
Role of CaM and CaMKII in [Ca2⫹]i Oscillations
As described in previous studies by our group (24, 45, 56),
1 ␮M ACh generated localized [Ca2⫹]i oscillations that propagate through the ASM cell. On ACh exposure, oscillation
frequency was high and oscillation amplitude was low. With
time, oscillation frequency decreased but amplitude increased,
such that peak [Ca2⫹]i was essentially unchanged. For data
Statistical Analysis
Experiments were performed using ASM cells obtained from at
least three tracheas. At least 10 cells were used per protocol. It was not
possible to apply all the experimental protocols to every cell. Differences between groups were assessed using one- or two-way ANOVA
as appropriate (with agonist, drug, and siRNA as variables). Post hoc
analyses were performed using Tukey’s test. For repeated measures,
Bonferroni’s corrections were applied. Statistical significance was
tested at P ⬍ 0.05.
AJP-Lung Cell Mol Physiol • VOL
Fig. 1. Inhibition of Ca2⫹ uptake in porcine airway smooth muscle (ASM) by
inhibition of calmodulin (CaM) via W-7 and CaM kinase (CaMKII) via KN-93 or
KN-62. Sarcoplasmic reticulum (SR) membranes isolated from porcine trachea
were incubated in the presence or absence of 10 ␮M antagonist, and Ca2⫹ uptake
was evaluated over a 30-min period. Values (means ⫾ SE) are expressed relative
to mean control Ca2⫹ uptake (n ⫽ 4 for W-7 and KN-62 and n ⫽ 5 for KN-93).
All values are significantly different from control: *P ⬍ 0.05.
294 • APRIL 2008 •
www.ajplung.org
Downloaded from http://ajplung.physiology.org/ by 10.220.32.247 on June 18, 2017
Effect of CaMKII Inhibition on PLN
L790
CALMODULIN AND SMOOTH MUSCLE Ca2⫹
P ⬍ 0.05). These effects were less than those of W-7 (Fig. 4A;
P ⬍ 0.05). Preexposure to KN-62 significantly increased basal
[Ca2⫹]i. Subsequent exposure to ACh resulted in a decrease in
[Ca2⫹]i oscillation frequency compared with control (i.e., the
same cell before ACh exposure; Fig. 4D; P ⬍ 0.05). However,
oscillation amplitude was largely unaffected.
Downloaded from http://ajplung.physiology.org/ by 10.220.32.247 on June 18, 2017
Fig. 2. Phospholamban (PLN) phosphorylation in porcine ASM. Relative
amounts of PLN phosphorylation in SR membranes isolated from porcine
trachea were evaluated under control (no Ca2⫹ or CaM), Ca2⫹/CaM, and
inhibited CaMKII conditions with use of phosphorylation site-specific [Ser16
(PSer-16-PLN) and Thr17 (PThr-17-PLN)] antibodies. A: relative amounts [arbitrary units (AU)] of PSer-16-PLN vs. PThr-17-PLN. B: representative immunoblots. SERCA2, sarco(endo)plasmic reticulum Ca2⫹-ATPase isoform 2. Values are
means ⫾ SE from 4 separate preparations. *Significantly different from unstimulated control (P ⬍ 0.05). #Significant effect of CaMKII inhibition (P ⬍ 0.05).
analysis purposes, only the steady-state oscillations (once the
trough and frequency are stable) were chosen. In control
experiments, removal of ACh (and washing of cells) followed
by reexposure to ACh resulted in recurrence of [Ca2⫹]i oscillations with amplitude and frequency similar to the first exposure. Removal of extracellular Ca2⫹ and addition of La3⫹ did
not significantly affect oscillation parameters under steadystate conditions.
During [Ca2⫹]i oscillations, exposure to the CaM antagonist
W-7 completely abolished the oscillations or dampened the
amplitude and frequency of the oscillations (Fig. 3, A and B).
In cells where oscillations were not abolished, W-7 significantly increased the normalized fall time but significantly
decreased oscillation amplitude and frequency compared with
control values (Fig. 4A; P ⬍ 0.05). In other experiments,
preexposure to 25 ␮M W-7 (n ⫽ 17 cells) followed by ACh (in
the continued presence of W-7) significantly increased trough
[Ca2⫹]i and fall time (P ⬍ 0.05) of [Ca2⫹]i oscillations and
decreased peak amplitude and frequency compared with ACh
exposure alone in the same cells (Fig. 4B; P ⬍ 0.05). In some
cells, ACh exposure in the presence of W-7 did not result in
[Ca2⫹]i oscillations.
In contrast to W-7, the CaMKII inhibitor KN-62 did not
abolish ongoing ACh-induced [Ca2⫹]i oscillations (Fig. 3C).
However, exposure to KN-62 significantly decreased oscillation amplitude and frequency and increased fall time (Fig. 4C;
AJP-Lung Cell Mol Physiol • VOL
Fig. 3. Effect of CaM and CaMKII inhibition on ACh-induced intracellular
Ca2⫹ ([Ca2⫹]i) oscillations in ASM cells. In enzymatically dissociated porcine
ASM cells, 1 ␮M ACh resulted in [Ca2⫹]i oscillations in the presence of
0-Ca2⫹ Hanks’ balanced salt solution (HBSS) and 1 mM La3⫹, thus representing repetitive SR Ca2⫹ release and reuptake (45). W-7 inhibited ongoing
oscillations (A) and decreased oscillation amplitude and frequency (B). KN-62
(CaMKII antagonist) slowed oscillation amplitude and frequency (C), but not
to the same extent as W-7.
294 • APRIL 2008 •
www.ajplung.org
CALMODULIN AND SMOOTH MUSCLE Ca2⫹
L791
In ASM cells that displayed a frequently described transient
[Ca2⫹]i response to 1 ␮M ACh (Fig. 5A), this response was
reproducible in control cells after washout over 20 –30 min
followed by reexposure to ACh. However, exposure to KN-62
or KN-93 before a second ACh exposure significantly slowed
the rate of fall of the [Ca2⫹]i transient (quantified by the time
constant via a single-exponential curve fit) compared with
controls (Fig. 5B; P ⬍ 0.05). This effect was persistent in the
presence and absence of extracellular Ca2⫹.
To confirm this concept, we performed additional experiments using the siRNA technique to knock down PLN and
studied the Ca2⫹ transients in response to ACh with and
without KN-93. The efficacy of siRNA knockdown was verified by
Western analysis of decreased PLN expression (Fig. 6A). Compared with cells exposed to Lipofectamine 2000 alone (vehicle
control), siRNA suppression of PLN resulted in a significantly
lower time constant for decay of [Ca2⫹]i (i.e., faster decline) in
the absence of extracellular Ca2⫹, confirming the role of PLN
in regulation of SERCA on Ca2⫹ uptake (Fig. 6C; P ⬍ 0.05).
Exposure to KN-93 significantly slowed the rate of [Ca2⫹]i
decline, even in PLN siRNA-transfected cells.
Role of CaM and CaMKII in [Ca2⫹]i Response to Caffeine
In ASM cells exposed to 5 mM caffeine, we observed the
characteristic transient [Ca2⫹]i response, even in 0-Ca2⫹ HBSS
with La3⫹. The peak response to caffeine was similar, even in
the absence of La3⫹, emphasizing the caffeine-mediated release of Ca2⫹ from the SR. Compared with control, 25 ␮M
W-7 significantly decreased the fall time of the [Ca2⫹]i response to caffeine but did not significantly affect the peak of
the response. However, basal [Ca2⫹]i was elevated in the
presence of W-7 (Fig. 7). Similar to W-7, in the presence of
KN-62, the peak [Ca2⫹]i response to caffeine was unaffected,
but fall time was significantly increased (Fig. 7; P ⬍ 0.05).
AJP-Lung Cell Mol Physiol • VOL
DISCUSSION
The results of the present study demonstrate that SR Ca2⫹
reuptake in ASM cells involves modulation by the CaMCaMKII pathway, with a role for the normally inhibitory
protein PLN. ACh-induced [Ca2⫹]i oscillations, which are
known to represent repetitive SR Ca2⫹ release and reuptake,
are shown to be modulated by CaM and/or CaMKII.
SR Ca2⫹ Reuptake in ASM
Although several intracellular mechanisms exist for sequestering Ca2⫹ in ASM (and, indeed, other smooth muscle cells),
including the SR, mitochondria, lysosomes, and nuclear envelope (22), it is generally accepted that SR Ca2⫹ stores are key
to regulation of ASM contractility. In this regard, the only
major mechanism that directly controls replenishment of SR
Ca2⫹ stores is SERCA. Given the extremely high (⬃10,000fold) Ca2⫹ gradient across the SR membrane, ATP hydrolysis
is required for transport of Ca2⫹ into the SR. Two Ca2⫹ are
pumped back into the SR for every ATP consumed. Indeed,
SERCA plays a key role in maintaining resting Ca2⫹ levels,
since inhibitors such as cyclopiazonic acid and thapsigargin
have been shown to increase [Ca2⫹]i (which supports the idea
of a constant SR leak, presumably via IP3 and RyR channels).
Inhibition of SERCA depletes SR Ca2⫹ stores, blunting the
[Ca2⫹]i response to agonist. Thus regulation of SERCA is a
key aspect of [Ca2⫹]i regulation not only in ASM, but in other
cell types as well.
Regulation of SR Ca2⫹ Reuptake
In cardiac muscle, it is well established that SERCA is
modulated in an inhibitory fashion by the protein PLN (60).
Previous studies in vascular smooth muscle have shown that,
as in cardiac muscle, phosphorylation and dephosphorylation
294 • APRIL 2008 •
www.ajplung.org
Downloaded from http://ajplung.physiology.org/ by 10.220.32.247 on June 18, 2017
Fig. 4. Effect of CaM and CaMKII inhibition on
ACh-induced [Ca2⫹]i oscillations in ASM cells. W-7
(A) and KN-62 (C) decreased oscillation amplitude and
frequency and increased fall time (FT) of individual
oscillations (normalized for oscillation amplitude). Exposure to W-7 (B) or KN-62 (D) before ACh application additionally resulted in elevation of basal [Ca2⫹]i
levels (especially with KN-62). Effects of either antagonist on oscillation amplitude and frequency were
greater during oscillations. *Significantly different
from control (P ⬍ 0.05). #Significant difference between W-7 and KN-62 (P ⬍ 0.05).
L792
CALMODULIN AND SMOOTH MUSCLE Ca2⫹
decline during electrical stimulation is increased, as are the
rates of contraction and relaxation (54). Interestingly, studies
of PLN in smooth muscle are not conclusive. In bladder
smooth muscle, SR Ca2⫹ load is increased and the rate of
[Ca2⫹]i decline is increased in PLN-knockout mice (42). Furthermore, in cardiac muscle, skeletal muscle, and several
smooth muscle types, SERCA activity and SR Ca2⫹ load are
increased by CaMII-mediated PLN phosphorylation (12, 42).
In contrast, in coronary artery smooth muscle, there is evidence
of CaMKII phosphorylation of SERCA with an accompanying
increase in SERCA activity, even though PLN could not be
detected in that tissue (18). In aortic smooth muscle, although
PLN protein was detected, no CaMKII-mediated phosphorylaDownloaded from http://ajplung.physiology.org/ by 10.220.32.247 on June 18, 2017
Fig. 5. Effect of CaMKII inhibition on decay of ACh-induced [Ca2⫹]i transients in ASM cells. A: typical recordings of [Ca2⫹]i transients. B: exposure to
KN-62 resulted in a higher time constant for decay of [Ca2⫹]i (i.e., slower
decline) in the presence and absence of extracellular Ca2⫹. HBSS, Hanks’
balanced salt solution. Values are means ⫾ SE. *Significantly different from
control (P ⬍ 0.05).
of PLN regulate SERCA activity, such that phosphorylation of
PLN by cyclic nucleotides, Ca2⫹/CaM kinase, and/or PKC
results in disinhibition of SERCA and accelerated SR Ca2⫹
reuptake (11, 49 –51). Furthermore, siRNA-mediated inhibition of PLN expression has been shown to improve Ca2⫹
uptake affinity and activity (65). Whether PLN is involved in
regulating SERCA activity in ASM has been examined in only
one study by McGraw et al. (38), where gene and protein
analysis of cultured ASM cells from transgenic mice overexpressing ␤2-adrenoceptors demonstrated decreased PLN levels,
correlating with markedly reduced responses of ASM tissue to
methacholine challenge. However, the bronchodilatory effect
of ␤-adrenoceptor stimulation was unaffected by the absence
of PLN. Therefore, the dynamic role of PLN in SR Ca2⫹
regulation was not defined. Interestingly, no data beyond those
presented by McGraw et al. specifically show PLN expression
in ASM. In this regard, the data provided in the present study
are novel. Furthermore, our data demonstrating that PLN
siRNA transfection yields a significantly faster decline in
[Ca2⫹]i indicate increased activity of SERCA and confirm the
regulatory role of PLN on Ca2⫹ uptake in ASM.
Studies in striated muscle have demonstrated that CaM (via
CaM kinases) can regulate SERCA via PLN (41). For example,
in cardiac myocytes of PLN-knockout mice, the rate of [Ca2⫹]i
AJP-Lung Cell Mol Physiol • VOL
Fig. 6. Effect of small interference RNA (siRNA) knockdown of PLN on
ACh-induced [Ca2⫹]i responses. Knockdown of PLN was verified by Western
analysis (A and B). Actin served as a loading control. PLN siRNA accelerated
decay of ACh-induced [Ca2⫹]i transients in the absence of extracellular Ca2⫹,
indicating that PLN has a role in regulation of SERCA Ca2⫹ uptake (C). Under
these conditions, CaMKII inhibition slowed reuptake, suggesting a direct
effect on SERCA. Values are means ⫾ SE. *Significant effect of PLN (P ⬍
0.05). #Significant effect of KN93 in PLN siRNA groups (P ⬍ 0.05).
294 • APRIL 2008 •
www.ajplung.org
CALMODULIN AND SMOOTH MUSCLE Ca2⫹
L793
CaM in [Ca2⫹]i Regulation
2⫹
Fig. 7. Effect of CaM and CaMKII inhibition on [Ca ]i response to caffeine.
In the presence of W-7 or KN-62, fall time of the characteristic transient
[Ca2⫹]i response of ASM cells to 5 mM caffeine was significantly increased
but peak of [Ca2⫹]i response was largely unaffected. Basal [Ca2⫹]i levels were
significantly elevated. *Significantly different from control (P ⬍ 0.05).
tion was found (8). In ASM, there is no evidence for PLN
regulation of SERCA. However, previous studies in SR vesicles from bovine trachea have shown that SERCA is modulated
by a Ca2⫹/CaM protein kinase (10). The results of the present
study not only demonstrate that PLN is present in porcine ASM
but, also, that CaM and CaMKII regulate SERCA potentially
by PLN (as evidenced by the increased Thr17 phosphorylation
of PLN, which was blocked by CaMKII inhibition). An interesting observation in this regard was the persistent Ser16
phosphorylation of PLN (which was unaffected by CaMKII
inhibition). Studies in cardiac muscle have established that
␤-adrenergic stimulation or other mechanisms that activate
PKA result in PLN phosphorylation at Ser16 and Thr17 (9,
13–15, 35, 36); however, the extent of Ser16 phosphorylation
appears to be greater and more consistent with PKA effects.
Although the present study did not specifically control for
inadvertent elevation of PKA during manipulation of samples,
the role of this phosphorylation site remains to be examined.
[Ca2⫹]i Oscillations in ASM
Although Ca2⫹ influx and SR Ca2⫹ release play important
roles in the [Ca2⫹]i response of ASM to agonist, we previously
AJP-Lung Cell Mol Physiol • VOL
CaM is a ubiquitous Ca2⫹-binding protein that activates
numerous substrates in the presence of increased [Ca2⫹]i,
including CaM kinase. On Ca2⫹/CaM binding, CaM kinase
undergoes autophosphorylation, leading to full enzyme activation (26, 29). W-7 has been reported to inhibit CaM activation
by selectively binding to CaM in a Ca2⫹-dependent manner
(20), whereas KN-62 or KN-93 inhibits CaMKII by blocking
the Ca2⫹/CaM-binding site on the enzyme, but not the ATPbinding domain (21).
In addition to the well-studied role of CaM in binding to
Ca2⫹ resulting in activation of smooth muscle myosin light
chain kinase and smooth muscle cell contraction (for review
see Refs. 17 and 64), CaM has also been found to modulate
several other [Ca2⫹]i regulatory mechanisms. In this regard,
the roles of CaM and CaM kinases in Ca2⫹ regulation differ
between striated and smooth muscles, as well as within smooth
muscle types. One of the effects studied in several cell systems
is the direct CaM regulation of the plasma membrane Ca2⫹ATPase (for review see Refs. 7 and 58). In non-ASM, plasma
membrane Ca2⫹-ATPase has been shown to at least contribute
to [Ca2⫹]i regulation (34, 55). Accordingly, CaM regulation of
the plasma membrane Ca2⫹-ATPase would have been a confounding issue in the present study on ASM. However, the use
of zero extracellular Ca2⫹ and La3⫹ to functionally inhibit the
plasma membrane likely minimized the contribution of CaM
regulation of the plasma membrane Ca2⫹-ATPase in our experiments. Accordingly, it is likely that the CaM (and CaMKII)
effects observed in the present study reflect interactions at the
level of the SR.
294 • APRIL 2008 •
www.ajplung.org
Downloaded from http://ajplung.physiology.org/ by 10.220.32.247 on June 18, 2017
found that ACh-induced [Ca2⫹]i oscillations reflect all-or-none
SR Ca2⫹ release from IP3-independent stores via RyR channels
but require SR Ca2⫹ release through IP3 receptor channels for
initiation (24, 45, 56). Furthermore, we demonstrated that
oscillation amplitude and frequency are modulated by ACh
concentration (47). However, we also found that although
blocking Ca2⫹ influx (e.g., 0-Ca2⫹ HBSS) eventually prevented the sustained steady-state phase of oscillations, the
oscillations were sustained in the presence of zero extracellular
Ca2⫹ and La3⫹ (which blocked Ca2⫹ influx and efflux). Finally, ACh-induced Ca2⫹ oscillations were observed in ␤-escin-permeabilized ASM cells (24). These results indicated that
ACh-induced [Ca2⫹]i oscillations arise from SR Ca2⫹ release
and that Ca2⫹ influx is required only to replenish the SR Ca2⫹
stores. Accordingly, in the present study, the use of zero
extracellular Ca2⫹ and La3⫹ allowed us to examine CaM
regulation of the SR. In our model of [Ca2⫹]i oscillations, the
amplitude of each oscillation represents the size of the SR
Ca2⫹ pool (SR Ca2⫹ content), and oscillation frequency reflects the sensitivity for SR Ca2⫹ release, including that for
Ca2⫹ release via RyR channels, which is itself affected by
[Ca2⫹]i levels (i.e., sensitivity to Ca2⫹-induced Ca2⫹ release).
The rise time of an individual oscillation reflects the rate of SR
Ca2⫹ release (which is dependent on the gradient for Ca2⫹
release) and the fall time the rate of reuptake (in the absence of
plasma membrane Ca2⫹ fluxes). Thus, under the conditions of
the experiment, the fall time of [Ca2⫹]i oscillations should
represent SR Ca2⫹ reuptake.
L794
CALMODULIN AND SMOOTH MUSCLE Ca2⫹
CaM, CaMKII, and SR Ca2⫹ Reuptake
The extent of SR Ca2⫹ release in response to agonist is
dependent on luminal SR Ca2⫹ stores, as well as the gradient
for release. In this regard, [Ca2⫹]i oscillations represent a
frequency-modulated response of cells to agonist stimulation,
allowing for increasing mean [Ca2⫹]i by increasing oscillation
frequency via a fixed SR Ca2⫹ store (43, 45, 56). Accordingly, factors that influence the extent or the sensitivity of
SR Ca2⫹ release will affect the amplitude and frequency of
[Ca2⫹]i oscillations and, thus, the ability of cells to respond
to agonist.
On the basis of studies in skeletal and cardiac muscles, CaM
regulation of SR Ca2⫹ release appears to be extremely complex
and dynamic, with several other factors affecting the net effect
on [Ca2⫹]i regulation (for review see Ref. 4). CaM binds to IP3
receptors (66) and RyR channels (3, 16), albeit with less
avidity for IP3 receptors. At low [Ca2⫹]i, CaM appears to
stimulate Ca2⫹ release via RyR channels or to have no effect
but does inhibit release at higher [Ca2⫹]i (39, 63). Previous
studies have also reported that cyclic ADP-ribose-mediated SR
Ca2⫹ release [which is thought to involve RyR channels (24,
46)] requires the presence of Ca2⫹-CaM (31, 32, 61, 62).
Although these complex effects of CaM are RyR isoform
specific, these results are relevant to ASM, where we previously showed the existence of all three RyR isoforms (24). In
our model of [Ca2⫹]i oscillations, where repetitive SR Ca2⫹
release occurs via RyR channels at steady state, CaM modulation of RyR channels can be expected to alter the sensitivity
for release (i.e., Ca2⫹-induced Ca2⫹ release) and, thus, the
amplitude and frequency of oscillations. In this regard, the
results of the present study of W-7-induced decreased oscillation amplitude and frequency are consistent.
Interestingly, we found that, in addition to W-7, KN-62 also
altered oscillation amplitude and frequency. Although direct
CaM regulation (with or without Ca2⫹) of RyR channels has
been demonstrated, less is known about the role of CaMKII. In
cardiac myocytes, endogenous CaMKII activity modulates SR
Ca2⫹ cycling during excitation-contraction coupling (33).
There is no information on CaMKII regulation of smooth
muscle RyR; however, the present results are suggestive.
In contrast to the ACh data, we found that the peak [Ca2⫹]i
response to caffeine was largely unaffected by W-7 or KN-62.
It is possible that the stimulatory effect of caffeine on SR Ca2⫹
release via RyR channels overwhelmed any modulatory effect
of CaM or CaMKII inhibition on these channels.
In our model of [Ca2⫹]i oscillations, SR Ca2⫹ release via IP3
receptor channels is required at least for initiation of oscillations. Previous studies have shown that CaM inhibits release
via these channels (40, 44). Accordingly, during oscillations,
when release via IP3 channels may be less important, CaM
inhibition by W-7 may have minimal effect. However, with
exposure to W-7 before ACh application, CaM may increase
basal Ca2⫹ release via IP3 receptor channels. This would lead
to increased [Ca2⫹]i, as observed in this study. The effects of
CaM and CaMKII inhibition on ongoing oscillations may
involve effects at IP3 receptor channels; however, with the
protocols used in the present study, it is difficult to isolate such
effects from concurrent changes in RyR channel activity.
CaM inhibition by W-7 and CaMKII inhibition by KN-62
resulted in significant slowing of the fall time of [Ca2⫹]i
oscillations in ASM cells in 0 Ca2⫹ and La3⫹, indicating a
slower reuptake. Furthermore, the fall time of the [Ca2⫹]i
response to caffeine was also significantly slowed in the
presence of these antagonists. Although the CaMKII inhibitor
KN-62 also decreased the above-mentioned parameters of
[Ca2⫹]i oscillations and the response to caffeine, these effects
were less pronounced than those of W-7. Nevertheless, the
effects of KN-62 suggest that the CaM regulation of SR Ca2⫹
reuptake is mediated via the kinase.
We also observed that baseline [Ca2⫹]i was significantly
elevated by W-7 and, especially, by KN-62 (even in the
absence of ACh). This may represent relative inhibition of
SERCA with ongoing SR Ca2⫹ leak. Furthermore, baseline
Ca2⫹ release via IP3 and RyR channels may be altered, resulting in a net increase in [Ca2⫹]i. Further study is needed to
examine these issues.
In conclusion, the present study demonstrates that, in porcine ASM, SR Ca2⫹ reuptake is modulated by CaM and/or
CaMKII, likely via concurrent effects on PLN and on release
via RyR channels.
AJP-Lung Cell Mol Physiol • VOL
ACKNOWLEDGMENTS
The authors gratefully acknowledge the assistance of Thomas Keller.
GRANTS
This work was supported by National Institutes of Health Grants HL-74309
(G. C. Sieck) and 1 UL1 RR-024150-01 (Mayo Clinic Clinical Research
awards to C. M. Pabelick and Y. S. Prakash from the National Center for
Research Resources) and by a Mayo Clinic Early Career Development Award
to Y. S. Prakash.
REFERENCES
1. Ay B, Prakash YS, Pabelick CM, Sieck GC. Store-operated Ca2⫹ entry
in porcine airway smooth muscle. Am J Physiol Lung Cell Mol Physiol
286: L909 –L917, 2004.
2. Bai Y, Sanderson MJ. Airway smooth muscle relaxation results from a
reduction in the frequency of Ca2⫹ oscillations induced by a cAMPmediated inhibition of the IP3 receptor. Respir Res 7: 34, 2006.
3. Balshaw DM, Xu L, Yamaguchi N, Pasek DA, Meissner G. Calmodulin
binding and inhibition of cardiac muscle calcium release channel (ryanodine receptor). J Biol Chem 276: 20144 –20153, 2001.
4. Balshaw DM, Yamaguchi N, Meissner G. Modulation of intracellular
calcium-release channels by calmodulin. J Membr Biol 185: 1– 8, 2002.
5. Bhupathy P, Babu GJ, Periasamy M. Sarcolipin and phospholamban as
regulators of cardiac sarcoplasmic reticulum Ca2⫹ ATPase. J Mol Cell
Cardiol 42: 903–911, 2007.
6. Brading AF. The sarcoplasmic reticulum in disease and smooth muscle
dysfunction: therapeutic potential. Novartis Found Symp 246: 244 –254,
2002.
7. Carafoli E, Garcia-Martin E, Guerini D. The plasma membrane calcium pump: recent developments and future perspectives. Experientia 52:
1091–1100, 1996.
8. Chen W, Lah M, Robinson PJ, Kemp BE. Phosphorylation of phospholamban in aortic smooth muscle cells and heart by calcium/calmodulin-dependent protein kinase II. Cell Signal 6: 617– 630, 1994.
9. Chu G, Kranias EG. Phospholamban as a therapeutic modality in heart
failure. Novartis Found Symp 274: 156 –171, 2006.
10. De Alfonzo RG, de Becemberg IL, Alfonzo MJ. A Ca2⫹/CAM protein
kinase associated with Ca2⫹ transport in sarco(endo)plasmic vesicles from
tracheal smooth muscle. Life Sci 58: 1403–1412, 1996.
11. Ferguson DG, Franzini-Armstrong C. The Ca2⫹-ATPase content of
slow and fast twitch fibers of guinea pig. Muscle Nerve 11: 561–570, 1988.
294 • APRIL 2008 •
www.ajplung.org
Downloaded from http://ajplung.physiology.org/ by 10.220.32.247 on June 18, 2017
CaM and SR Ca2⫹ Release
CALMODULIN AND SMOOTH MUSCLE Ca2⫹
AJP-Lung Cell Mol Physiol • VOL
36. Mattiazzi A, Mundina-Weilenmann C, Vittone L, Said M, Kranias
EG. The importance of the Thr17 residue of phospholamban as a phosphorylation site under physiological and pathological conditions. Braz
J Med Biol Res 39: 563–572, 2006.
37. Mattiazzi A, Vittone L, Mundina-Weilenmann C. Ca2⫹/calmodulindependent protein kinase: a key component in the contractile recovery
from acidosis. Cardiovasc Res 73: 648 – 656, 2007.
38. McGraw DW, Fogel KM, Kong S, Litonjua AA, Kranias EG, Aronow
BJ, Liggett SB. Transcriptional response to persistent ␤2-adrenergic
receptor signaling reveals regulation of phospholamban, which alters
airway contractility. Physiol Genomics 27: 171–177, 2006.
39. Meissner G. Evidence of a role for calmodulin in the regulation of
calcium release from skeletal muscle sarcoplasmic reticulum. Biochemistry 25: 244 –251, 1986.
40. Missiaen L, Parys JB, Weidema AF, Sipma H, Vanlingen S, De Smet
P, Callewaert G, De Smedt H. The bell-shaped Ca2⫹ dependence of the
inositol 1,4,5-trisphosphate-induced Ca2⫹ release is modulated by Ca2⫹/
calmodulin. J Biol Chem 274: 13748 –13751, 1999.
41. Narayanan N, Xu A. Phosphorylation and regulation of the Ca2⫹pumping ATPase in cardiac sarcoplasmic reticulum by calcium/calmodulin-dependent protein kinase. Basic Res Cardiol 92 Suppl 1: 25–35, 1997.
42. Nobe K, Sutliff RL, Kranias EG, Paul RJ. Phospholamban regulation of
bladder contractility: evidence from gene-altered mouse models. J Physiol
535: 867– 878, 2001.
43. Pabelick CM, Sieck GC, Prakash YS. Significance of spatial and
temporal heterogeneity of calcium transients in smooth muscle. J Appl
Physiol 91: 488 – 496, 2001.
44. Patel S, Joseph SK, Thomas AP. Molecular properties of inositol
1,4,5-trisphosphate receptors. Cell Calcium 25: 247–264, 1999.
45. Prakash YS, Kannan MS, Sieck GC. Regulation of intracellular calcium
oscillations in porcine tracheal smooth muscle cells. Am J Physiol Cell
Physiol 272: C966 –C975, 1997.
46. Prakash YS, Kannan MS, Walseth TF, Sieck GC. Role of cyclic
ADP-ribose in the regulation of [Ca2⫹]i in porcine tracheal smooth
muscle. Am J Physiol Cell Physiol 274: C1653–C1660, 1998.
47. Prakash YS, Pabelick CM, Kannan MS, Sieck GC. Spatial and temporal aspects of ACh-induced [Ca2⫹]i oscillations in porcine tracheal
smooth muscle. Cell Calcium 27: 153–162, 2000.
48. Prakash YS, Thompson MA, Vaa B, Matabdin I, Peterson TE, He T,
Pabelick CM. Caveolins and intracellular calcium regulation in human
airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 293: L1118 –
L1123, 2007.
49. Raeymaekers L, Eggermont JA, Wuytack F, Casteels R. Effects of
cyclic nucleotide dependent protein kinases on the endoplasmic reticulum
Ca2⫹ pump of bovine pulmonary artery. Cell Calcium 11: 261–268, 1990.
50. Raeymaekers L, Hofmann F, Casteels R. Cyclic GMP-dependent protein kinase phosphorylates phospholamban in isolated sarcoplasmic reticulum from cardiac and smooth muscle. Biochem J 252: 269 –273, 1988.
51. Raeymaekers L, Jones LR. Evidence for the presence of phospholamban
in the endoplasmic reticulum of smooth muscle. Biochim Biophys Acta
882: 258 –265, 1986.
52. Sanders KM. Mechanisms of calcium handling in smooth muscles. J Appl
Physiol 91: 1438 –1449, 2001.
53. Sathish V, Xu A, Karmazyn M, Sims SM, Narayanan N. Mechanistic
basis of differences in Ca2⫹-handling properties of sarcoplasmic reticulum
in right and left ventricles of normal rat myocardium. Am J Physiol Heart
Circ Physiol 291: H88 –H96, 2006.
54. Schmidt AG, Edes I, Kranias EG. Phospholamban: a promising therapeutic target in heart failure? Cardiovasc Drugs Ther 15: 387–396, 2001.
55. Schuh K, Quaschning T, Knauer S, Hu K, Kocak S, Roethlein N,
Neyses L. Regulation of vascular tone in animals overexpressing the
sarcolemmal calcium pump. J Biol Chem 278: 41246 – 41252, 2003.
56. Sieck GC, Kannan MS, Prakash YS. Heterogeneity in dynamic regulation of intracellular calcium in airway smooth muscle cells. Can J Physiol
Pharmacol 75: 878 – 888, 1997.
57. Simmerman HK, Jones LR. Phospholamban: protein structure, mechanism of
action, and role in cardiac function. Physiol Rev 78: 921–947, 1998.
58. Strehler EE, Zacharias DA. Role of alternative splicing in generating
isoform diversity among plasma membrane calcium pumps. Physiol Rev
81: 21–50, 2001.
59. Sutliff RL, Conforti L, Weber CS, Kranias EG, Paul RJ. Regulation of
the spontaneous contractile activity of the portal vein by the sarcoplasmic
reticulum: evidence from the phospholamban gene-ablated mouse. Vasc
Pharmacol 41: 197–204, 2004.
294 • APRIL 2008 •
www.ajplung.org
Downloaded from http://ajplung.physiology.org/ by 10.220.32.247 on June 18, 2017
12. Ferrington DA, Yao Q, Squier TC, Bigelow DJ. Comparable levels of
Ca-ATPase inhibition by phospholamban in slow-twitch skeletal and
cardiac sarcoplasmic reticulum. Biochemistry 41: 13289 –13296, 2002.
13. Frank K, Kranias EG. Phospholamban and cardiac contractility. Ann
Med 32: 572–578, 2000.
14. Frank KF, Bolck B, Brixius K, Kranias EG, Schwinger RH. Modulation of SERCA: implications for the failing human heart. Basic Res
Cardiol 97 Suppl I: I72–I78, 2002.
15. Frank KF, Bolck B, Erdmann E, Schwinger RH. Sarcoplasmic reticulum Ca2⫹-ATPase modulates cardiac contraction and relaxation. Cardiovasc Res 57: 20 –27, 2003.
16. Fruen BR, Black DJ, Bloomquist RA, Bardy JM, Johnson JD, Louis
CF, Balog EM. Regulation of the RYR1 and RYR2 Ca2⫹ release channel
isoforms by Ca2⫹-insensitive mutants of calmodulin. Biochemistry 42:
2740 –2747, 2003.
17. Gerthoffer WT. Regulation of the contractile element of airway smooth
muscle. Am J Physiol Lung Cell Mol Physiol 261: L15–L28, 1991.
18. Grover AK, Xu A, Samson SE, Narayanan N. Sarcoplasmic reticulum
Ca2⫹ pump in pig coronary artery smooth muscle is regulated by a novel
pathway. Am J Physiol Cell Physiol 271: C181–C187, 1996.
19. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2⫹
indicators with greatly improved fluorescence properties. J Biol Chem
260: 3440 –3450, 1985.
20. Hidaka H, Yamaki T, Naka M, Tanaka T, Hayashi H, Kobayashi R.
Calcium-regulated modulator protein interacting agents inhibit smooth
muscle calcium-stimulated protein kinase and ATPase. Mol Pharmacol
17: 66 –72, 1980.
21. Hidaka H, Yokokura H. Molecular and cellular pharmacology of a
calcium/calmodulin-dependent protein kinase II (CaM kinase II) inhibitor,
KN-62, and proposal of CaM kinase phosphorylation cascades. Adv
Pharmacol 36: 193–219, 1996.
22. Hirota S, Helli P, Janssen LJ. Ionic mechanisms and Ca2⫹ handling in
airway smooth muscle. Eur Respir J 30: 114 –133, 2007.
23. Hisayama T, Takayanagi I. Ryanodine: its possible mechanism of action
in the caffeine-sensitive calcium store of smooth muscle. Pflügers Arch
412: 376 –381, 1988.
24. Kannan MS, Prakash YS, Brenner T, Mickelson JR, Sieck GC. Role
of ryanodine receptor channels in Ca2⫹ oscillations of porcine tracheal
smooth muscle. Am J Physiol Lung Cell Mol Physiol 272: L659 –L664,
1997.
25. Karczewski P, Hendrischke T, Wolf WP, Morano I, Bartel S,
Schrader J. Phosphorylation of phospholamban correlates with relaxation
of coronary artery induced by nitric oxide, adenosine, and prostacyclin in
the pig. J Cell Biochem 70: 49 –59, 1998.
26. Katoh T, Fujisawa H. Autoactivation of calmodulin-dependent protein
kinase II by autophosphorylation. J Biol Chem 266: 3039 –3044, 1991.
27. Kim M, Han IS, Koh SD, Perrino BA. Roles of CaM kinase II and
phospholamban in SNP-induced relaxation of murine gastric fundus
smooth muscles. Am J Physiol Cell Physiol 291: C337–C347, 2006.
28. Kim M, Perrino BA. CaM kinase II activation and phospholamban
phosphorylation by SNP in murine gastric antrum smooth muscles. Am J
Physiol Gastrointest Liver Physiol 292: G1045–G1054, 2007.
29. Kwiatkowski AP, Shell DJ, King MM. The role of autophosphorylation
in activation of the type II calmodulin-dependent protein kinase. J Biol
Chem 263: 6484 – 6486, 1988.
30. Lalli MJ, Shimizu S, Sutliff RL, Kranias EG, Paul RJ. [Ca2⫹]i
homeostasis and cyclic nucleotide relaxation in aorta of phospholambandeficient mice. Am J Physiol Heart Circ Physiol 277: H963–H970, 1999.
31. Lee HC, Aarhus R, Graeff R, Gurnack ME, Walseth TF. Cyclic ADP
ribose activation of the ryanodine receptor is mediated by calmodulin.
Nature 370: 307–309, 1994.
32. Lee HC, Aarhus R, Graeff RM. Sensitization of calcium-induced calcium release by cyclic ADP-ribose and calmodulin. J Biol Chem 270:
9060 –9066, 1995.
33. Li L, Satoh H, Ginsburg KS, Bers DM. The effect of Ca2⫹-calmodulindependent protein kinase II on cardiac excitation-contraction coupling in
ferret ventricular myocytes. J Physiol 501: 17–31, 1997.
34. Matthew A, Shmygol A, Wray S. Ca2⫹ entry, efflux and release in
smooth muscle. Biol Res 37: 617– 624, 2004.
35. Mattiazzi A, Mundina-Weilenmann C, Guoxiang C, Vittone L, Kranias E. Role of phospholamban phosphorylation on Thr17 in cardiac
physiological and pathological conditions. Cardiovasc Res 68: 366 –375,
2005.
L795
L796
CALMODULIN AND SMOOTH MUSCLE Ca2⫹
60. Tada M, Katz AM. Phosphorylation of the sarcoplasmic reticulum and
sarcolemma. Annu Rev Physiol 44: 401– 423, 1982.
61. Takasawa S, Ishida A, Nata K, Nakagawa K, Noguchi N, Tohgo A,
Kato I, Yonekura H, Fujisawa H, Okamoto H. Requirement of calmodulin-dependent protein kinase II in cyclic ADP-ribose-mediated intracellular Ca2⫹ mobilization. J Biol Chem 270: 30257–30259, 1995.
62. Tanaka Y, Tashjian AJ. Calmodulin is a selective mediator of Ca2⫹induced Ca2⫹-release via ryanodine receptor-like Ca2⫹-channel triggered
by cyclic ADP ribose. Proc Natl Acad Sci USA 92: 3244 –3248, 1995.
63. Tripathy A, Xu L, Mann G, Meissner G. Calmodulin activation and
inhibition of skeletal muscle Ca2⫹ release channel (ryanodine receptor).
Biophys J 69: 106 –119, 1995.
64. Walsh MP. Calcium-dependent mechanisms of regulation of smooth
muscle contraction. Biochem Cell Biol 69: 771– 800, 1991.
65. Watanabe A, Arai M, Yamazaki M, Koitabashi N, Wuytack F,
Kurabayashi M. Phospholamban ablation by RNA interference increases
Ca2⫹ uptake into rat cardiac myocyte sarcoplasmic reticulum. J Mol Cell
Cardiol 37: 691– 698, 2004.
66. Yamada M, Miyawaki A, Saito K, Nakajima T, Yamamoto-Hino M, Ryo
Y, Furuichi T, Mikoshiba K. The calmodulin-binding domain in the mouse
type 1 inositol 1,4,5-trisphosphate receptor. Biochem J 308: 83– 88, 1995.
67. Yusufi AN, Cheng J, Thompson MA, Burnett JC, Grande JP. Differential mechanisms of Ca2⫹ release from vascular smooth muscle cell
microsomes. Exp Biol Med (Maywood) 227: 36 – 44, 2002.
Downloaded from http://ajplung.physiology.org/ by 10.220.32.247 on June 18, 2017
AJP-Lung Cell Mol Physiol • VOL
294 • APRIL 2008 •
www.ajplung.org