Am J Physiol Lung Cell Mol Physiol 307: L781–L790, 2014.
First published September 19, 2014; doi:10.1152/ajplung.00149.2014.
Calcineurin upregulates local Ca2⫹ signaling through ryanodine receptor-1 in
airway smooth muscle cells
Carlo P. Savoia,1 Qing-Hua Liu,1,2 Yun-Min Zheng,1 Vishal Yadav,1 Zhen Zhang,3 Ling-Gang Wu,3
and Yong-Xiao Wang1
1
Center for Cardiovascular Sciences, Albany Medical College, Albany, New York; 2Institute for Medical Biology, College of
Life Sciences, South-Central University for Nationalities, Wuhan, Hubei, China; 3Synaptic Transmission Section, National
Institute of Neurological Disorders and Stroke, Bethesda, Maryland
Submitted 6 June 2014; accepted in final form 18 September 2014
local calcium signaling; contraction
hypercontractile responses to nonspecific stimuli of airway smooth muscle cells (ASMCs) encircling the respiratory tree, pose a life-threatening risk to ⬎300
million people worldwide on a daily basis. The pathological
airway hyperresponsiveness (AHR) has long been thought to
be associated with dysfunctional Ca2⫹-signaling pathways involved in the regulation of ASMC contraction.
Ca2⫹ signaling may occur in the form of local Ca2⫹ release
events such as a Ca2⫹ spark, which is due to the synchronized
opening of a cluster of ryanodine receptors (RyRs), the main
intracellular Ca2⫹ release channels on the sarcoplasmic reticulum (SR) (6, 7). Ca2⫹ sparks have been observed in equine
ASTHMA ATTACKS, DUE TO
Address for reprint requests and other correspondence: Y.-X. Wang, Albany
Medical College, Center for Cardiovascular Sciences (MC-8), 47 New Scotland Ave., Albany, NY 12208 (e-mail: [email protected]).
http://www.ajplung.org
(44), guinea pig (51), porcine (32), mouse (26), and human
ASMCs (10). These local Ca2⫹ events are known to regulate
ASMC membrane potential through the activation of hyperpolarizing spontaneous transient outward currents (STOCs) or
depolarizing spontaneous transient inward currents (STICs)
(49). Ca2⫹ sparks can cause STOCs by opening big-conductance Ca2⫹-activated K⫹ (BK) channels that are located nearby
RyRs in ASMCs, leading to K⫹ efflux, membrane hyperpolarization, and a decrease in the sensitivity of ASMCs to excitation (20, 22). Oppositely, Ca2⫹ sparks activate STICs due to a
close association of RyRs with highly dense clusters of Ca2⫹activated Cl⫺ (CaCl) channels (1) that have now been identified
as the transmembrane protein 16A CaCl channel (5, 36, 46). Of
great clinical importance in determining the cause of asthmatic
AHR, Ca2⫹ sparks have recently been shown to exist in human
ASMCs (10) and to be upregulated in freshly isolated ASMCs
from a sensitized murine model of asthma (41). Thus, Ca2⫹
sparks may play an important role in physiological and pathological functions in ASMCs.
We have reported that a direct activation of native M3
muscarinic receptors following exposure of the receptor agonist methacholine (MCh) or membrane depolarization results
in activation of a Gq protein-coupled receptor-mediated signaling cascade, initiating the phospholipase C (PLC)-dependent
breakdown of phosphatidylinositol 4,5-bisphosphate into diacylgycerol (DAG) and inositol 1,4,5-trisphosphate (IP3); IP3
activates IP3 receptors (IP3Rs), inducing intracellular Ca2⫹
release; this activates nearby RyR2 via a local Ca2⫹-induced
Ca2⫹-release (CICR) mechanism, amplifying agonist-evoked,
IP3R-mediated Ca2⫹ release and contraction in ASMCs (25).
As a result of the MCh exposure or membrane depolarization,
PLC also produces DAG, which activates protein kinase C
(PKC). It has been shown that PKC downregulates Ca2⫹
sparks in vascular smooth muscle cells (SMCs) (3). Our study
has shown that PKC⑀ inhibits Ca2⫹ sparks specifically through
RyR1 and MCh-induced contraction in ASMCs (24). Because
a kinase is generally believed to regulate an ion channel in
coordination with one or more complementary phosphatases, a
protein phosphatase may promote RyR1-mediated local Ca2⫹
signals and contraction in ASMC. The serine/threonine protein
phosphatase 2B (calcineurin, CaN) has been shown to regulate
Ca2⫹ release in a C2C12 cell line derived from mouse skeletal
muscle, indicating its role in the control of RyR1 activity (37).
It is believed that CaN has a regulatory effect and potential
physical interaction with intracellular Ca2⫹ release channels
(4). CaN has also been shown to oppositely modulate PKC
substrates like the transient receptor potential vanilloid-1
(TRPV1) channels in chorda tympani taste neurons (28), the
1040-0605/14 Copyright © 2014 the American Physiological Society
L781
Downloaded from http://ajplung.physiology.org/ by 10.220.32.247 on June 14, 2017
Savoia CP, Liu QH, Zheng YM, Yadav V, Zhang Z, Wu LG,
Wang YX. Calcineurin upregulates local Ca2⫹ signaling through
ryanodine receptor-1 in airway smooth muscle cells. Am J Physiol
Lung Cell Mol Physiol 307: L781–L790, 2014. First published September 19, 2014; doi:10.1152/ajplung.00149.2014.—Local Ca2⫹ signals (Ca2⫹ sparks) play an important role in multiple cellular functions in airway smooth muscle cells (ASMCs). Protein kinase C⑀ is
known to downregulate ASMC Ca2⫹ sparks and contraction; however, no complementary phosphatase has been shown to produce
opposite effects. Here, we for the first time report that treatment with
a specific calcineurin (CaN) autoinhibitory peptide (CAIP) to block
CaN activity decreases, whereas application of nickel to activate CaN
increases, Ca2⫹ sparks in both the presence and absence of extracellular Ca2⫹. Treatment with xestospogin-C to eliminate functional
inositol 1,4,5-trisphosphate receptors does not prevent CAIP from
inhibiting local Ca2⫹ signaling. However, high ryanodine treatment
almost completely blocks spark formation and prevents the nickelmediated increase in sparks. Unlike CAIP, the protein phosphatase 2A
inhibitor endothall has no effect. Local Ca2⫹ signaling is lower in
CaN catalytic subunit A␣ gene knockout (CaN-A␣⫺/⫺) mouse
ASMCs. The effects of CAIP and nickel are completely lost in
CaN-A␣⫺/⫺ ASMCs. Neither CAIP nor nickel produces an effect on
Ca2⫹ sparks in type 1 ryanodine receptor heterozygous knockout
(RyR1⫺/⫹) mouse ASMCs. However, their effects are not altered in
RyR2⫺/⫹ or RyR3⫺/⫺ mouse ASMCs. CaN inhibition decreases
methacholine-induced contraction in isolated RyR1⫹/⫹ but not RyR1⫺/⫹
mouse tracheal rings. Supportively, muscarinic contractile responses are
also reduced in CaN-A␣⫺/⫹ mouse tracheal rings. Taken together, these
results provide novel evidence that CaN regulates ASMC Ca2⫹ sparks
specifically through RyR1, which plays an important role in the control of
Ca2⫹ signaling and contraction in ASMCs.
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CALCINEURIN UPREGULATES LOCAL Ca2⫹ SIGNALING IN AIRWAY MYOCYTES
myristoylated alanine-rich C kinase substrate (MARCKS), and
growth-associated protein43 (GAP43) in the rat hippocampus
(47) and L-type Ca2⫹ channels in vascular SMCs (30). Taken
together, in the current study, we performed a series of pharmacological and genetic studies to test a novel hypothesis that
CaN is a suitable phosphatase of interest in the regulation of
local Ca2⫹ signaling and associated contraction in ASMCs,
and the role of CaN may also be specifically mediated by
RyR1.
MATERIALS AND METHODS
RESULTS
Specific inhibition of CaN decreases ASMC local Ca2⫹
signaling. Specific inhibition of CaN was achieved by treatment with the synthetic CAIP (20 ␮M). Ca2⫹ sparks were first
recorded in freshly isolated ASMCs before treatment as control. After that, cells were exposed to CAIP, which was
delivered by a pressure injection via a micropippette connected
to a Picospritzer III system, as described in our previous
publication (43). Following exposure of CAIP for 8 min, Ca2⫹
sparks were recorded again. Due to the cell, animal, and
day-to-day variation, the frequencies and amplitudes of Ca2⫹
sparks were normalized to those before treatment with CAIP
(control) and analyzed using a paired Student’s t-test to determine their statistically significant differences. As shown in Fig.
1A, CAIP treatment reduced local Ca2⫹ signaling, decreasing
the spontaneous Ca2⫹ spark frequency by 58 ⫾ 8% (n ⫽ 11,
P ⬍ 0.05; Fig. 1B). However, the amplitude of Ca2⫹ sparks
was not significantly changed. In control experiments, we
found that a pressure injection of normal bath solution for 8
min had no effect on either Ca2⫹ spark frequency or amplitude
in eight cells tested.
After seeing this, we wanted to verify that the effect of CAIP
on local Ca2⫹ signaling was due to intracellular Ca2⫹ release,
rather than extracellular Ca2⫹ influx. Thus, we repeated the
initial experiments with cells bathed in nominally free Ca2⫹
PSS supplemented with 0.5 mM EGTA to scavenge extracellular Ca2⫹. Highlighted in Fig. 1C, we saw that CAIP de-
AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00149.2014 • www.ajplung.org
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Cell isolation. Freshly isolated mouse ASMCs were prepared as
previously reported (24 –26). In brief, adult male Swiss Webster mice
were killed by an intraperitoneal injection of pentobarbital sodium
according to the protocol approved by the Animal Care and Use
Committee of Albany Medical College. The trachea was removed and
kept in ice-cold physiological saline solution (PSS) containing (in
mM): 125 NaCl, 5 KCl, 1 MgSO4, 10 glucose, 10 HEPES, and 1.8
CaCl2 (pH 7.4). The tissue was then cleaned of epithelium, cartilage,
and connective tissue and then digested using a two-step enzymatic
method. First the trachealis muscle tissue strip was incubated in low
(0.1 mM)-Ca2⫹ PSS (37°C) containing (in mg/ml): 1 papain, 0.2
dithioerythritol, and 1 bovine serum albumin (BSA) for ⬃12 min and
then in low-Ca2⫹ PSS containing (in mg/ml): 1 dithiothreitol, 1
collagenase H, 1 collagenase II, and 1 BSA for ⬃27 min. The tissue
was washed four times for 5 min with 5 ml of low-Ca2⫹ PSS
containing 1 mg/ml BSA and then gently triturated to release single
cells for daily use (⬃6 h) in high-Ca2⫹ PSS.
Animals. Swiss Webster male mice at an age of 6 – 8 wk were
purchased from Taconic Farms (Rensselaer, NY). RyR1 heterozygous, RyR2 heterozygous, and RyR3 homozygous gene knockout
mice (RyR1⫺/⫹, RyR2⫺/⫹, and RyR3⫺/⫺) of C57/BL6 background
were originally provided by Dr. Takeshima at Kyoto University
Graduate School of Pharmaceutical Sciences (Kyoto, Japan) and bred
to produce RyR1⫺/⫹, RyR2⫺/⫹, and RyR3⫺/⫺ mice and their corresponding wild-type (RyR1⫹/⫹, RyR2⫹/⫹, and RyR3⫹/⫹) animals, as
described in our previous publications (24 –26). C57/BL6-based CaN
catalytic subunit A␣ homozygous knockout (CaN A␣⫺/⫺) mice were
offered by Dr. J. L. Gooch at Emory University School of Medicine
(Atlanta, GA) and bred to generate CaN A␣⫺/⫺, CaN A␣⫺/⫹, and
CaN A␣⫹/⫹ mice, as we reported previously (39). RyR1⫺/⫺ mice die
at or just before birth; RyR2⫺/⫺ mice are embryonically lethal; and
RyR1⫺/⫹, RyR2⫺/⫹, RyR3⫺/⫺, CaN-A␣⫺/⫺, as well as CaN-A␣⫺/⫹
mice show normal growth, viability, fertility, and behaviors and do
not have noticeable mortality or morbidity. However, CaN A␣ KO
mice are rarely born and have only been provided by Dr. Zhang and
Dr. Wu at the National Institute of Neurological Disorders and Stroke
(NINDS, Bethesda, MD). Thus, male RyR1⫺/⫹, RyR2⫺/⫹, RyR3⫺/⫺,
CaN A␣⫺/⫺, CaN A␣⫺/⫹, and their wild-type mice at an age of 6 –10
wk were used in experiments.
Ca2⫹ imaging. Spontaneous local Ca2⫹ signaling (Ca2⫹ sparks)
was measured in freshly isolated ASMCs using an LSM510 laserscanning confocal microscope (Carl Zeiss) (26). Cells were loaded
with fluo 4-AM (2.5 ␮M) in 1.8 mM Ca2⫹ PSS at room temperature
for 30 min and then perfused with PSS to rinse away excess fluo
4-AM. Images were taken using a line-scanning mode with a Zeiss
⫻40 oil immersion objective. Fluo 4 was excited at 488 nm, and
emitted fluorescence was measured at 505 nm. The confocal pinhole
was set at 1 airy unit to provide a spatial resolution of 0.9 ␮m in the
x-y axis. Each line-scan image was taken every 1.9 ms. The original
line-scan recording times before and after application of agents were
set at 5 s to obtain a number of Ca2⫹ sparks and minimize laser
toxicity.
Muscle contraction. Muscle contraction in isolated tracheal rings
was measured using the organ bath technique, as described previously
(44), with isometric transducers (Harvard Apparatus, South Natick,
MA) and a PowerLab/4SP recording system (AD Instruments, Colorado Springs, CO). Tracheas were quickly removed from mice and
transferred into ice-cold 1.8 mM PSS. After the connective tissue and
epithelia were removed, tracheal rings were mounted vertically in
2-ml organ bath chambers containing Krebs solution (in mM): 110
NaCl, 3.4 KCl, 2.4 CaCl2, 0.8 MgSO4, 25.8 NaHCO3, 1.2 KH2PO4,
and 5.6 glucose (equilibrated with 20% O2, 5% CO2, and 75% N2, pH
7.4) at 37°C. The resting tension was set at 500 mg. During a 60-min
equilibration period, rings were washed every 20 min and restretched
to 500 mg. The muscarinic receptor agonist MCh was given to induce
muscle contraction.
Reagents. CaN autoinhibitory peptide (CAIP), xestospongin-C
(Xes-C), and phorbol 12-myristate,13-acetate were purchased from
Calbiochem. Fluo 4-AM was purchased from Molecular Probes.
These chemicals were dissolved in water or dimethyl sulfoxide with a
final concentration ⱕ0.1%. Nickel chloride, MCh, and other remaining reagents were purchased from Sigma. The Calcineurin Cellular
Activity Assay Kit was purchased from Enzolifesciences (catalog no.
BML-AK816 – 0001). Pharmacological agents were delivered on the
cell through a glass pipette connected to a Picospritzer III pressure
controller (Parker Instrumentation) or directly added by pipette to the
organ tissue bath solution. All experiments were conducted at room
temperature (⬃22°C). Data were obtained from a minimum of three
different animals.
Statistical analysis. All of the data are presented as means ⫾ SE.
Statistical comparisons in the same cells before and after treatment
with pharmacological agents were performed using the paired Student’s t-test and normalized to control. Statistical comparisons between different mouse genotypes were performed using an unpaired
Student’s t-test. Statistical analysis of isolated tracheal rings before
and after treatment was done using a paired Student’s t-test. A
statistical comparison between contractile responses in different
mouse genotypes was performed using an unpaired Student’s t-test.
Differences with a P value ⬍0.05 were considered statistically significant.
CALCINEURIN UPREGULATES LOCAL Ca2⫹ SIGNALING IN AIRWAY MYOCYTES
A
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2⫹
Fig. 1. Specific inhibition of calcineurin (CaN) decreases local Ca signaling
in mouse airway smooth muscle cells (ASMCs). A: original recordings exhibit
local Ca2⫹ signals (sparks) in a mouse ASMC before (control) and after
treatment with a specific CaN autoinhibitory peptide (CAIP, 20 ␮M) for 8 min.
Ca2⫹ sparks were recorded using a Zeiss LSM510 laser-scanning confocal
microscope with a line-scanning mode. B: bar graphs summarize the effects of
CAIP on the frequency and amplitude of Ca2⫹ sparks. Nos. in parentheses
indicate the no. of individual cells tested from a minimum of three mice. *P ⬍
0.05 compared with control (before application of CAIP). C: effect of CAIP
treatment on Ca2⫹ sparks in ASMCs in the absence of extracellular Ca2⫹
(nominally free Ca2⫹ PSS supplemented with 0.5 mM EGTA). D: effects of
CAIP on the frequency and amplitude of Ca2⫹ sparks in ASMCs pretreated
with xestongin-C (Xes-C, 10 ␮M) for 8 min to functionally inhibit inositol
1,4,5-trisphosphate receptors (IP3Rs). E: effects of the protein phosphatase 2A
endothall (1 ␮M) for 8 min on local Ca2⫹ signaling in cells pretreated with
Xes-C (10 ␮M) for 8 min.
creased local Ca2⫹ signaling by over 50% in the absence of
extracellular Ca2⫹ with no effect on amplitude, similar to those
in the presence of extracellular Ca2⫹. These results suggest that
CaN promotes Ca2⫹ sparks through intracellular Ca2⫹ release
pathways.
We have previously shown that IP3Rs promote local Ca2⫹
release events in ASMCs through a local CICR mechanism
(24 –26), and so the action of a phosphatase, which increases
Ca2⫹ sparks, could function through the IP3R-mediated CICR
process. To exclude this possible complication, we tested the
effects of CaN inhibition (via CAIP) on Ca2⫹ sparks in the
absence of functional IP3Rs. In the current study, the functional
elimination of IP3Rs was obtained by pretreating cells with
Xes-C at 10 ␮M for 8 min, as we described previously (26).
Ca2⫹ sparks were measured in cells pretreated with Xes-C as
control; then, the cells were treated with CAIP (20 ␮M) for 8
min, and Ca2⫹ sparks were recorded. Figure 1D shows that
CAIP still decreased local Ca2⫹ signals even in cells pretreated
with Xes-C, signifying that CaN might upregulate local Ca2⫹
signaling by regulating RyRs in ASMCs.
To confirm the specific effect of CaN in general, we tested
whether inhibition of protein phosphatase 2A (PP2A) might
have a similar effect. In Fig. 1E, cells were pretreated with
Xes-C to functionally inhibit IP3Rs, and then endothall was
applied at 1 ␮M for 8 min to inhibit PP2A. Endothall had no
effect on Ca2⫹ spark frequency or amplitude, suggesting a
specific and novel role for the serine/threonine protein phosphatase CaN in promoting local Ca2⫹ signaling in ASMCs
through its effect on RyRs.
Pharmacological activation of CaN increases local Ca2⫹
signaling in ASMCs. Because CaN inhibition decreases local
Ca2⫹ signaling, we next examined whether CaN activation
might lead to an increase in local Ca2⫹ signals. The activation
of CaN was achieved by treatment with nickel, which has been
shown to activate purified CaN from bovine brain and bacteria
(18, 33, 34). In contrast to CAIP, application of nickel at 500
␮M for 5 min greatly increased the mean frequency of Ca2⫹
sparks by 110 ⫾ 25% (n ⫽ 13; P ⬍ 0.05; Fig. 2A), whereas the
mean amplitude of Ca2⫹ sparks was unaltered.
Our subsequent experiment was to test whether nickel could
increase sparks by interacting with CaN following treatment
with CAIP. In these experiments, Ca2⫹ sparks were recorded in
cells before and after the successive treatment of CAIP (20 ␮M)
for 8 min followed by nickel (500 ␮M) for 5 min (13 min total
between scans). Our results in Fig. 2C show no change in local
Ca2⫹ signaling before and after the dual treatment, signifying
that nickel was able to reverse the expected decrease in sparks
following CAIP (seen in Fig. 1), restoring local Ca2⫹ signaling
to control levels and further supporting the claim that nickel
may increase Ca2⫹ sparks by activating CaN. We double
checked the effect of nickel on Ca2⫹ sparks in the absence of
extracellular Ca2⫹ and found that nickel was still able to
significantly increase local Ca2⫹ signals even in the absence of
extracellular Ca2⫹ (Fig. 2D).
Subsequently, we wanted to verify that RyR-mediated Ca2⫹
release is indeed responsible for Ca2⫹ sparks and that RyRs are
required for the CaN-based regulation of local Ca2⫹ signaling.
We treated spontaneously sparking mouse ASMCs with ryanodine (100 ␮M) for 7 min, followed by nickel (500 ␮M) for 5
min. As summarized in Fig. 2E, a high concentration of
ryanodine treatment almost completely blocked all sparks in all
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Frequency
(% control)
B
L783
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CALCINEURIN UPREGULATES LOCAL Ca2⫹ SIGNALING IN AIRWAY MYOCYTES
cells, as was also seen in a previous report (26), and currently
ryanodine treatment prevented nickel from generating any
further Ca2⫹ release in the form of sparks. These results
illustrate that RyRs are indeed responsible for Ca2⫹ spark
formation and that CaN requires them to regulate sparks.
As a control experiment, to confirm that the time between
scans for various treatments has no effect on sparking cells, we
A
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Fig. 2. Pharmacological activation of CaN increases local Ca2⫹ signaling in
ASMCs. A: original recordings exhibit local Ca2⫹ signals (sparks) in a mouse
ASMC before (control) and after treatment with nickel (500 ␮M) for 5 min. B:
bar graphs summarize the effects of nickel on the frequency and amplitude of
Ca2⫹ sparks. Nos. in parentheses indicate the no. of individual cells tested
from a minimum of three mice. *P ⬍ 0.05 compared with control (before
application of nickel). C: effects of CAIP (20 ␮M) for 8 min followed by
nickel (500 ␮M) for 5 min on the frequency and amplitude of ASMC Ca2⫹
sparks. D: effect of nickel treatment on Ca2⫹ sparks in ASMCs in the
absence of extracellular Ca2⫹ (nominally free Ca2⫹ PSS supplemented with
0.5 mM EGTA). E: effect of ryanodine (100 ␮M) for 7 min and then nickel
on the frequency and amplitude of ASMC Ca2⫹ sparks. F: effect of 1.8 mM
Ca2⫹ PSS for 8 and 13 min on the frequency and amplitude of local Ca2⫹
signaling in mouse ASMCs.
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0.5 ΔF/Fo
10 μm
Nickel
analyzed sparks before and after 8 and 13 min of 1.8 mM Ca2⫹
PSS. As summarized in Fig. 2F, there was no difference in
spark frequency or amplitude throughout a 13-min timespan
with saline treatment. Together, our studies provide clear
evidence that CaN may upregulate local Ca2⫹ signaling in
ASMCs.
Genetic inhibition of CaN decreases local Ca2⫹ signaling in
ASMCs. To further verify this role for CaN, we tested whether
genetic inhibition of CaN changed spontaneous local Ca2⫹
signaling. We used CaN-A␣⫺/⫺ mice because the CaN-A␣
domain has been shown to play an important role in controlling
the activity of CaCl channels and ATP-sensitive K⫹ channels in
SMCs (15, 31). Freshly isolated ASMCs were prepared in
parallel, from tracheal tissue obtained from CaN-A␣⫹/⫹ and
CaN-A␣⫺/⫺ mice provided by Dr. Zhang and Dr. Wu at the
NINDS. Ca2⫹ sparks were measured in both cell types, and
their frequency and amplitudes were compared using an unpaired Student’s t-test. Figure 3A shows example recordings of
spontaneous local Ca2⫹ signaling in a CaN-A␣⫹/⫹ mouse
ASMC vs. CaN-A␣⫺/⫺. As summarized in Fig. 3B, the mean
frequency of Ca2⫹ sparks was 0.0409 ⫾ 0.00419
sparks·␮m⫺1·s⫺1 in CaN-A␣⫺/⫺ cells and 0.0552 ⫾ 0.00755
sparks·␮m⫺1·s⫺1 in CaN-A␣⫹/⫹, whereas the amplitude was
similar in both cell types, providing evidence that CaN-A␣⫺/⫺
impairs ASMC local Ca2⫹ signaling.
With access to tissue from these knockout animals, we were
able to verify the results of our pharmacological experiments
using CAIP or nickel. As seen in Fig. 3C, treatment with the
specific synthetic peptide CAIP, to inhibit CaN, did not affect
Ca2⫹ sparks in CaN-A␣⫺/⫺ ASMCs. Similarly, application of
nickel to activate CaN did not produce an effect in CaN-A␣⫺/⫺
cells either (Fig. 3D). Collectively, the effects of CAIP and
nickel on local Ca2⫹ signaling in ASMCs result from their
specific inhibition and activation of CaN, respectively, and
local Ca2⫹ signaling is reduced in CaN-A␣⫺/⫺ ASMCs.
RyR1 gene deletion blocks the role of CaN in the regulation
of local Ca2⫹ signaling in ASMCs. Because we have just
outlined the role for the serine/threonine protein phosphatase
CaN in regulating local Ca2⫹ signaling, we questioned if it
works through RyR1. First, RyR1 heterozygous gene deletion
(RyR1⫺/⫹) mice were used because RyR1 homozygous knockout mice (RyR1⫺/⫺) die before or at birth; second, local Ca2⫹
signaling is significantly different in embryonic vs. adult
ASMCs; and third, the effects of PKC⑀ are fully blocked in
RyR1⫺/⫹ ASMCs (24, 25). Here, we found that treatment with
CAIP was without effect on local Ca2⫹ signals in RyR1⫺/⫹
CALCINEURIN UPREGULATES LOCAL Ca2⫹ SIGNALING IN AIRWAY MYOCYTES
A
CaN-Aα-/-
0.5 ΔF/Fo
0.5 ΔF/Fo
5 μm
5 μm
CaN-Aα+/+
1s
0.02
125
(n = 8)
Amplitude
(% control)
50
25
CaN-Aα+/+ CaN-Aα-/-
150
100
50
0
(n = 14)
75
50
25
CaN-Aα-/-
0.25
CAIP
100
0
0.50
200
75
CaN-Aα-/-
0.75
0.00
CaN-Aα+/+ CaN-Aα-/-
100
125
1.00
CAIP
CaN-Aα-/-
Nickel
100
75
50
25
0
Nickel
CaN-Aα-/-
125
Amplitude
(% control)
Frequency
(% control)
Frequency
(% control)
*
0.04
0
D
(n = 38)
0.06
0.00
C
(n = 16)
0.08
Amplitude
(ΔF/F0)
0.10
Fig. 3. Genetic inhibition of CaN decreases spontaneous local Ca2⫹ and
prevents the effects of pharmacological inhibition and activation. A: original
recordings exhibit local Ca2⫹ signals (sparks) in a CaN-A␣ wild-type (WT)
mouse compared with a CaN-A␣⫺/⫺ mouse ASMC. B: bar graphs present the
mean frequency and amplitude of Ca2⫹ sparks from CaN-A␣ WT and
CaN-A␣⫺/⫺ mouse ASMCs. Nos. in parentheses indicate the no. of individual
cells tested from a minimum of three mice. *P ⬍ 0.05 comparing CaN-A␣ WT
and CaN-A␣⫺/⫺. C: effect of CAIP (20 ␮M) for 8 min on Ca2⫹ sparks in
CaN-A␣⫺/⫺ mouse ASMCs. D: effect of nickel on the frequency and amplitude of local Ca2⫹ signaling in CaN-A␣⫺/⫺ mouse ASMCs.
ASMCs (Fig. 4, A and B), whereas it significantly decreased
spark frequency in RyR1⫹/⫹ cells (Fig. 4C).
After that, we tested the effect of CaN activation in
RyR1⫺/⫹ ASMCs. Application of nickel to activate CaN did
not alter the frequency and amplitude of Ca2⫹ sparks in
RyR1⫺/⫹ cells (Fig. 4D), although it greatly increased Ca2⫹
spark frequency in RyR1⫹/⫹ cells (Fig. 4E), paralleling the
results of Fig. 2, A and B.
To further examine the potentially important role of RyR1 in
mediating CaN-based regulation of ASMC local Ca2⫹ signaling, we sought to determine if there is a difference in basal
local Ca2⫹ signaling between RyR1⫹/⫹ and RyR1⫺/⫹ ASMCs.
We prepared freshly isolated ASMCs from both RyR1⫹/⫹ and
RyR1⫺/⫹ mice in parallel and imaged spontaneously sparking
cells from both populations comparing their frequency and
amplitude with an unpaired t-test. The results summarized in
Fig. 4F reveal that local Ca2⫹ signaling is significantly higher
in RyR1⫹/⫹ (0.05122 ⫾ 0.00339 sparks·␮m⫺1·s⫺1) vs.
RyR1⫺/⫹ (0.04266 ⫾ 0.00383) mice. Although the amplitudes
are not significantly different, the P value for an unpaired t-test
approached ⬃0.06, suggesting that the amplitude of sparks
might also be lower in RyR1⫺/⫹. Altogether, our results reveal
that CaN upregulates local Ca2⫹ signals through its specific
effect on RyR1 in ASMCs and that RyR1 plays a critical role
in the regulation of local Ca2⫹ signaling.
CaN regulates local Ca2⫹ signals independent of RyR2 or
RyR3. Next, we examined whether RyR2 or RyR3 might also
mediate the role of CaN in controlling the activity of local
Ca2⫹ signaling. Different from the results seen in RyR1⫺/⫹
ASMCs (Fig. 4), RyR2 heterozygous gene deletion (RyR2⫺/⫹)
did not block the effect of CaN inhibition or activation through
the application of CAIP or nickel, respectively. Figure 5, A
and B, reveals that CAIP resulted in a significant decrease in
the activity of Ca2⫹ sparks in RyR2⫺/⫹, whereas Fig. 5C
shows that nickel significantly increased local Ca2⫹ signals
in RyR2⫺/⫹ mouse ASMCs.
Similar to RyR2⫺/⫹, RyR3⫺/⫺ gene knockout was unable to
prevent CaN inhibition and activation from decreasing and
increasing the activity of Ca2⫹ sparks, respectively. In Fig. 6,
A and B, application of CAIP significantly decreased spark
frequency in RyR3⫺/⫺ ASMCs (n ⫽ 12; P ⬍ 0.05), whereas in
Fig. 6C nickel significantly increased spark frequency in
RyR3⫺/⫺ cells (n ⫽ 21; P ⬍ 0.05). These data reinforce the
view that CaN upregulates local Ca2⫹ signaling in ASMCs
through its specific effect on RyR1.
CaN inhibition regulates muscarinic contraction in airway
muscle through RyR1. Having seen the novel role for CaN in
upregulating local Ca2⫹ signaling through RyR1, we hypothesized that inhibiting CaN might affect the physiological contractile response of airway muscle to the classical muscarinic
receptor agonist MCh. In Fig. 7A we tested the contraction of
isolated tracheal rings to MCh before and after the addition
of saline (control) or CAIP (20 ␮M) for 45 min. Application
of MCh (10 ␮M) resulted in repeated contractile responses
with similar amplitudes in isolated tracheal rings. In contrast, tracheal rings pretreated with CAIP exhibited a decreased contractile response.
To verify the effect of CAIP in airway smooth muscle
tissues, we incubated the isolated tracheal rings with CAIP at
20 ␮M or saline and then measured the activity of CaN in
tracheal tissues with a Calcineurin Cellular Assay Kit (Calbiochem). The results are shown in Fig. 7B, in which CaN activity
was significantly decreased in tracheal tissues after incubation
of CAIP. Thus, CAIP can steadily enter ASMCs in tracheal
tissues to produce an inhibitory effect on Ca2⫹ sparks and
contraction.
Furthermore, we wanted to see if RyR1 heterozygous gene
deletion could block the effects of CAIP on MCh-induced
contraction. In Fig. 7C, we repeated the same experiments in
Fig. 7A but with tracheal rings taken from RyR1⫺/⫹ mice. The
results of our work show no change in contraction with saline
or CAIP in RyR1⫺/⫹, supporting the hypothesis that CaN
upregulates local Ca2⫹ signaling and contraction through
RyR1 in airway smooth muscle.
Enhancing our understanding of the physiological contractile role of CaN, we assessed the effect of genetic inhibition of
CaN on MCh-induced contraction in isolated tracheal rings.
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Frequency
(sparks/μm/s)
B
L785
L786
CALCINEURIN UPREGULATES LOCAL Ca2⫹ SIGNALING IN AIRWAY MYOCYTES
A
RyR 2-/+
5 μm
CAIP
0.5 ΔF/Fo
Previous studies have revealed that CaN-A␣⫺/⫹ mice have
decreased protein expression and activity (12, 13); thus, we
thought it suitable to compare MCh-evoked contractile responses in CaN-A␣⫹/⫹ and CaN-A␣⫺/⫹ mouse tracheal rings.
As seen in Fig. 7D, CaN-A␣⫺/⫹ rings have a decreased
contractile response to 10 ␮M MCh vs. CaN-A␣⫹/⫹ tissues,
1s
B
0.5 ΔF/Fo
1s
(n = 26)
100
75
50
25
0
75
*
50
25
50
100
75
50
0
CAIP
RyR 2-/+
CAIP
*
200
150
100
-/+
RyR 2
Nickel
RyR 2-/+
Nickel
125
100
75
50
25
0
CAIP
supporting our novel hypothesis that CaN regulates ASMC
contraction through RyR1-based local Ca2⫹ signaling.
DISCUSSION
125
100
75
50
0
Nickel
(n = 14)
200
150
100
(n = 55)
(n = 48)
RyR1+/+
Nickel
RyR1+/+
RyR1-/+
100
*
0.04
0.02
RyR1-/+
Ca2⫹ sparks, localized transient Ca2⫹ release events due to
the coordinated opening of a cluster of RyRs in a functional
Ca2⫹ release unit (CRU) on the SR, are known to regulate
excitation-contraction coupling, membrane excitability, neurotransmitter release and secretion, cell proliferation and migration, and gene expression in a variety of cell types (23, 29).
We and other investigators have shown the presence of Ca2⫹
sparks in equine, guinea pig, porcine, and mouse ASMCs (23),
making them an active area of respiratory research. Within
ASMCs, these localized Ca2⫹ release events directly mediate a
50
0.8
Amplitude
(ΔF/F0)
Nickel
RyR1+/+
Nickel
150
0
RyR1+/+
0.06
RyR1-/+
200
*
Amplitude
(% control)
Frequency
(% control)
RyR1-/+
50
Frequency
(sparks/μm/sec)
25
Fig. 5. CaN regulates local Ca signals independent of RyR type 2. A: original
recordings exhibit local Ca2⫹ signals (sparks) in a RyR2⫺/⫹ mouse ASMC
before (control) and after treatment with CAIP (20 ␮M) for 8 min. B: bar
graphs summarize the effects of CAIP on the frequency and amplitude of Ca2⫹
sparks in RyR2⫺/⫹. Nos. in parentheses indicate the no. of individual cells
tested from a minimum of three mice. *P ⬍ 0.05 compared with control
(before CAIP). C: effects of nickel (500 ␮M) for 5 min on the frequency and
amplitude of Ca2⫹ sparks in RyR2⫺/⫹.
25
250
0.00
50
(n = 6)
250
CAIP
150
Amplitude
(% control)
Frequency
(% control)
(n = 19)
75
0.08
RyR 2-/+
300
0
125
RyR1+/+
25
F
25
75
0
100
0
*
50
50
150
CAIP
125
300
25
25
RyR1+/+
E
75
100
2⫹
Amplitude
(% control)
Frequency
(% control)
100
0
50
RyR1-/+
(n = 9)
125
150
75
CAIP
0
D
100
0
RyR1-/+
C
C
125
Frequency
(% control)
125
125
100
0
Amplitude
(% control)
Frequency
(% control)
B
(n = 9)
125
0.6
0.4
0.2
0.0
Fig. 4. Ryanodine receptor-1 (RyR1) gene deletion blocks the role of CaN in
the regulation of local Ca2⫹ signaling. A: original recordings exhibit local
Ca2⫹ signals (sparks) in a RyR1⫺/⫹ mouse ASMC before (control) and after
treatment with CAIP (20 ␮M) for 8 min. B: bar graphs summarize the effects
of CAIP on the frequency and amplitude of Ca2⫹ sparks in RyR1⫺/⫹. Nos. in
parentheses indicate the no. of individual cells tested from a minimum of three
mice. *P ⬍ 0.05 compared with control (before application of CAIP). C: effect
of CAIP on the frequency and amplitude of Ca2⫹ sparks in RyR1⫹/⫹. D: effect
of nickel (500 ␮M) for 5 min on the frequency and amplitude of Ca2⫹ sparks
in RyR1⫺/⫹. E: effect of nickel on the frequency and amplitude of Ca2⫹ sparks
in RyR1⫹/⫹. F: bar graphs represent the mean frequency and amplitude of
local Ca2⫹ signals from RyR1⫹/⫹ and RyR1⫺/⫹ mouse ASMCs.
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Frequency
(% control)
10 μm
CAIP
Amplitude
(% control)
RyR1-/+
Amplitude
(% control)
A
L787
CALCINEURIN UPREGULATES LOCAL Ca2⫹ SIGNALING IN AIRWAY MYOCYTES
A
-/-
RyR3
logical conditions, membrane depolarization causes a direct
activation of Gq protein-coupled M3 muscarinic receptors,
leading to intracellular Ca2⫹ release and contraction without
the involvement of LTCCs in ASMCs (25), signifying the
especially important role of intracellular Ca2⫹ release for
A
1s
75
*
25
250
(n=21)
Frequency
(% control)
25
RyR3-/-
100
50
B
75
50
25
RyR3-/-
(n=6)
400
*
300
200
100
0
100
0
Nickel
(n=7)
500
CAIP
125
*
-/RyR3
50
CAIP
150
0
75
0
RyR3-/-
200
100
nM Po4/μg protein
50
Tension (mg/mg)
to 10 μM MCh
100
0
C
125
(n = 13)
Amplitude
(% control)
125
CAIP
Nickel
0.100
(n=3)
(n=3)
Before
After
0.050
*
0.025
0.000
Control
Tension (mg/mg)
to 10 μM MCh
C
CAIP
-/+
RyR1
Saline
600
CAIP
(n=4)
(n=6)
500
400
300
200
100
0
Before
D
Tension (mg/mg)
to MCh
basal tone of contraction, with cell relaxation and contraction
following a decrease or increase in their activity, respectively.
These local Ca2⫹ signals are also able to regulate ASMC
membrane potential (23). RyRs on the SR can localize with
other local cytosolic or membrane-bound proteins/channels,
structurally forming membrane-membrane nanojunctions and
functionally communicating using Ca2⫹ concentration as a
currency (42). It has been generally accepted that RyRs may
highly colocalize with ClCa and BK channels in ASMCs. By
this unique micromachinery, RyR-mediated Ca2⫹ sparks readily activate these two channels to generate STICs and STOCs,
respectively. STICs lead to membrane depolarization, LTCC
activation, and extracellular Ca2⫹ influx; in contrast, STOCs
result in membrane hyperpolarization and LTCC inhibition,
thereby inhibiting extracellular Ca2⫹ influx (23, 29). However,
at resting membrane potential, Ca2⫹ sparks preferentially activate STICs, whereas at more positive membrane potential
STOCs are initiated (19). STICs have been shown to be
involved in ASMC contraction (16) and asthmatic AHR (48).
In human ASMCs, some believe the spark-STOC relationship
underlies relaxation of ASMCs following activation of bitter
taste receptors (10). Nevertheless, previous reports have shown
that ASMC contraction is not significantly affected by LTCC
blockers in isolated animal ASMCs and tissues, and clinical
studies indicate that LTCC blockers are ineffective in the
treatment of airway smooth muscle contraction and asthma (9,
14, 38). Our recent research demonstrates that, under physio-
After
0.075
2⫹
Fig. 6. CaN regulates local Ca signals independent of RyR type 3. A: original
recordings exhibit local Ca2⫹ signals (sparks) in a RyR3⫺/⫺ mouse ASMC
before (control) and after treatment with CAIP (20 ␮M) for 8 min. B: bar
graphs summarize the effect of CAIP on the frequency and amplitude of Ca2⫹
sparks in RyR3⫺/⫺. Nos. in parentheses indicate the no. of individual cells
tested from a minimum of three mice. *P ⬍ 0.05 compared with control
(before CAIP). C: effects of nickel (500 ␮M) for 5 min on the frequency and
amplitude of Ca2⫹ sparks in RyR3⫺/⫺.
Before
After
Before
0.1 μM
After
10 μM
600
500
(n=6)
(n=6)
400
*
300
200
100
0
CaN WT
CaN HE
CaN WT
CaN HE
Fig. 7. CaN inhibition regulates muscarinic contraction in airway muscle
through RyR1. A: muscle contraction induced by the muscarinic receptor
agonist methacholine at 10 ␮M was measured in isolated tracheal rings from
RyR1⫹/⫹ mice before and after treatment with saline or CAIP (20 ␮M) for 45
min. The results are expressed as mg tension/mg of tissue. Nos. in parentheses
indicate the no. of individual rings tested from a minimum of three mice. *P ⬍
0.05 compared with before saline or CAIP. B: CAIP inhibits the activity of
CaN in tracheal ring tissue. CaN activity was inhibited in tracheal ring tissues
incubated with CAIP at 20 ␮M for 45 min vs. saline as measured through the
CaN Cellular Activity Assay Kit (Enzolifesciences: catalog no. BML-AK8160001). Nos. in parentheses denote the no. of times the kit was run with four
mouse airway tissue strips being used each time. *P ⬍ 0.05 for CAIP vs.
control. C: methacholine (10 ␮M)-induced contraction in isolated tracheal
rings from RyR⫺/⫹ mice before and after saline or CAIP (20 ␮M) for 45 min.
D: methacholine (0.1 and 10 ␮M) induced contraction in isolated tracheal rings
from CaN WT and heterozygous gene deletion mice.
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Frequency
(% control)
B
+/+
RyR1
Saline
600
Amplitude
(% control)
0.5 ΔF/Fo
5 μm
CAIP
L788
CALCINEURIN UPREGULATES LOCAL Ca2⫹ SIGNALING IN AIRWAY MYOCYTES
clear evidence for the novel role of CaN in upregulating ASMC
local Ca2⫹ signaling.
Our earlier study (24) has demonstrated a specific role for
RyR1 in mediating the downregulation of local Ca2⫹ signals
by PKC⑀, leading us to hypothesize that CaN may function
through RyR1. Local Ca2⫹ signaling is significantly different
between embryonic and adult ASMCs, suggesting that different regulatory mechanisms are in play (24). Furthermore,
within a given SMC there are multiple spark-generating sites
where individual Ca2⫹ spark currents have been measured over
a wide range, suggesting the involvement of any number from
1 to 50 RyRs in a single CRU (50) with each CRU made up of
a heterogeneous mix of RyR1 and RyR2 (but perhaps not
RyR3). As we reported previously (24), the effect of PKC⑀ on
Ca2⫹ sparks is completely blocked in RyR1 heterozygous
deletion (RyR1⫺/⫹) ASMCs, which is similar to that in RyR1
homozygous gene deletion (RyR1⫺/⫺) cells. With all these
considerations, we have used RyR1⫺/⫹ mice to test our current
hypothesis. Our data reveal a specific role for RyR1 in mediating the CaN-based regulation of Ca2⫹ sparks since the effects
of chemical inhibition and activation of CaN are lost in
RyR1⫺/⫹ ASMCs (Fig. 4). As a consequence, RyR1 full
expression is required for the CaN-based regulation of local
Ca2⫹ signaling in ASMCs. In support, RyR1 is also required
for the Ca2⫹ spark formation in skeletal muscle cells (35),
depolarization-induced Ca2⫹ spark in cultured portal vein (8),
and embryonic bladder SMCs (11). RyR1⫺/⫹ ASMCs have a
lower level of local Ca2⫹ signaling, further indicating the
specific importance of RyR1 in this cell type. However, all
three known RyR subtypes (RyR1, RyR2, and RyR3) are
expressed in ASMCs (23). However, in support of the specific
role for RyR1 in mediating CaN-based regulation of Ca2⫹
sparks, we have found that the effects of CaN inhibition and
activation are not blocked in RyR2⫺/⫹ or RyR3⫺/⫺ ASMCs
(Figs. 5 and 6). The role of RyR2 in Ca2⫹ spark signaling has
been extensively studied in the heart, but we know the least
about the role of RyR3 in the generation of Ca2⫹ sparks in
SMCs. RyR3 gene knockout or knockdown does not alter local
Ca2⫹ signaling in bladder and portal vein SMCs (8, 17); yet, it
has been reported that the frequency of STOCs is augmented in
RyR3⫺/⫺ cerebral artery myocytes (27). This result has been
interpreted as evidence for the inhibitory role of RyR3 in the
development of Ca2⫹ sparks, since STOCs are generally
thought to be activated by Ca2⫹ sparks. However, the frequency of Ca2⫹ sparks in RyR3⫺/⫺ cerebral artery myocytes is
not significantly increased. Furthermore, we have shown that
resting and depolarization-induced Ca2⫹ sparks are not prevented in RyR3⫺/⫺ ASMCs (25) and that the downregulation
of local Ca2⫹ signaling by PKC⑀ is not blocked in RyR3⫺/⫺
cells either (24). These findings, taken together, clearly point
out that CaN regulates local Ca2⫹ signaling specifically
through RyR1, but not RyR2 or RyR3.
As previously stated, Ca2⫹ sparks play an essential role in
mediating ASMC contractile tone as well as regulating ASMC
excitation-contraction coupling through the activation of
STICs and STOCs. For the first time, we have shown how CaN
promotes airway muscle contraction in response to muscarinic
stimulation through RyR1-mediated local Ca2⫹ signaling. CaN
inhibition via CAIP decreases MCh-induced contraction in
RyR1⫹/⫹ rings (Fig. 7A), but not in RyR1⫺/⫹ rings (Fig. 7C).
In support, a similar smaller muscarinic contractile response
AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00149.2014 • www.ajplung.org
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ASMC contraction. Of great clinical importance in the context
of the aforementioned information is the fact that local Ca2⫹
signals have been shown to be significantly upregulated in a
mouse model of asthma (41). These findings lead us to believe
that an increase in local Ca2⫹ signals may perturb the dynamic
balance of STICs and STOCs, forming a mechanistic setting
for the exaggerated AHR seen in asthma. Thus, new regulators
of ASMC local Ca2⫹ signaling, once defined, may become
novel drug targets for the treatment of asthma.
Recent work from our laboratory has shown that PKC⑀
downregulates local Ca2⫹ signaling through its specific effect
on RyR1 as well as inhibiting the contractile response of
ASMCs to MCh (23). Because a kinase regulates the activity of
its substrate in coordination with one or more phosphatases by
causing phosphorylation and dephosphorylation, we wondered
whether and which phosphatase is involved in the regulation of
local Ca2⫹ signals in ASMCs. Protein phosphatase 2B/CaN
(CaN) is known to regulate RyR-mediated Ca2⫹ release in a
C2C12 cell line (37). Moreover, this serine/threonine protein
phosphatase, opposite to the serine/threonine protein kinase
PKC, controls the activity of TRPV1 channels (28) GAP43,
MARCKS (47), and LTCCs in vascular SMCs (30). Therefore,
we proposed a novel hypothesis that CaN may upregulate local
Ca2⫹ signaling and contraction in ASMCs. In support of this
view, we have found that specific inhibition of CaN with CAIP
reduces, whereas activation of CaN with nickel increases, local
Ca2⫹ signaling (Figs. 1A, 1B, 2A, and 2B). The inhibitory
effect of CaN in C2C12 cells is thought to depend on CaN, RyR,
and the 12-kDa FK-506-binding protein (FKBP 12) being
associated in a trimeric complex (37); however, within
ASMCs, FKBP 12 does not bind to RyRs (44), providing
support that CaN regulates ASMC RyR activity through a
novel regulatory mechanism. As shown in Fig. 1C, a loss of
extracellular Ca2⫹ influx (under nominally free extracellular
Ca2⫹ conditions with EGTA) has no effects on the decrease in
Ca2⫹ sparks due to CAIP; similarly, the effect of CaN activation with nickel is not affected either (Fig. 2D), suggesting that
extracellular Ca2⫹ is not necessary for the regulation of Ca2⫹
sparks by CaN. Within a murine model of asthma, ASMC local
Ca2⫹ signaling is increased (41); it is unknown how this
increase is maintained, and it might seem reasonable to suggest
that the canonical transient receptor potential-3 channel, which
mediates extracellular Ca2⫹ influx and has increased expression and activity in asthma (45), may play a role in maintaining
this signaling. We have further reinforced the specific role for
the protein phosphatase CaN by finding out that inhibition of
PP2A with endothall does not affect local Ca2⫹ signals (Fig.
1E), unlike in cardiac myocytes where PP2A was shown to
increase Ca2⫹ sparks (40). RyRs are essential for spark formation; however, IP3Rs cross talk with RyRs, promoting Ca2⫹
spark formation through a local CICR process (25, 26). In the
present study, we have revealed that CaN is able to modulate
Ca2⫹ sparks in the absence of functional IP3Rs (Fig. 1D) but
not in the absence of functional RyRs (Figs. 2E, 4A, 4B, and
4D). These results suggest that CaN regulates RyRs and then
local Ca2⫹ signaling in ASMCs.
More importantly, local Ca2⫹ signaling is decreased in
CaN-A␣⫺/⫺ ASMCs (Fig. 3), and both CAIP and nickel fail to
affect Ca2⫹ sparks in the knockout cells. These results indicate
that CAIP and nickel are specifically targeting CaN, providing
CALCINEURIN UPREGULATES LOCAL Ca2⫹ SIGNALING IN AIRWAY MYOCYTES
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
GRANTS
This work was supported by National Heart, Lung, and Blood Institute
Grant R01-HL-071000 (Y.-X. Wang), an American Heart Association Established Investigator Award 0340160N (Y.-X. Wang), as well as Scientist
Development Grants 0730242N (Q.-H. Liu) and 0630236N (Y.-M. Zheng).
17.
DISCLOSURES
No conflicts of interest, financial or otherwise are declared by the authors.
AUTHOR CONTRIBUTIONS
18.
19.
C.P.S., Q.-H.L., Y.-M.Z., and Y.-X.W. conception and design of research;
C.P.S., Q.-H.L., Y.-M.Z., V.R.Y., Z.Z., L.-G.W., and Y.-X.W. performed
experiments; C.P.S., Q.-H.L., Y.-M.Z., and V.R.Y. analyzed data; C.P.S.,
Q.-H.L., Y.-M.Z., V.R.Y., Z.Z., L.-G.W., and Y.-X.W. interpreted results of
experiments; C.P.S., Q.-H.L., Y.-M.Z., V.R.Y., and Y.-X.W. prepared Figs.;
C.P.S., Q.-H.L., Y.-M.Z., V.R.Y., and Y.-X.W. drafted manuscript; C.P.S.,
Q.-H.L., Y.-M.Z., V.R.Y., Z.Z., L.-G.W., and Y.-X.W. edited and revised
manuscript; C.P.S., Q.-H.L., Y.-M.Z., V.R.Y., Z.Z., L.-G.W., and Y.-X.W.
approved final version of manuscript.
20.
21.
22.
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CALCINEURIN UPREGULATES LOCAL Ca2⫹ SIGNALING IN AIRWAY MYOCYTES