From the Vascular Biology Unit, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts 02118
Received for publication, November 6, 2000, and in revised form, December 7, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Depletion of intracellular
Ca2+ stores activates capacitative Ca2+
influx in smooth muscle cells, but the native store-operated channels
that mediate such influx remain unidentified. Recently we demonstrated
that calcium influx factor produced by yeast and human platelets
with depleted Ca2+ stores activates small conductance
cation channels in excised membrane patches from vascular smooth muscle
cells (SMC). Here we characterize these channels in intact cells and
present evidence that they belong to the class of store-operated
channels, which are activated upon passive depletion of
Ca2+ stores. Application of thapsigargin (TG), an inhibitor
of sarco-endoplasmic reticulum Ca2+ ATPase, to individual
SMC activated single 3-pS cation channels in cell-attached membrane
patches. Channels remained active when inside-out membrane patches were
excised from the cells. Excision of membrane patches from resting SMC
did not by itself activate the channels. Load-ing SMC with BAPTA
(1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid), which slowly depletes Ca2+ stores without a rise in
intracellular Ca2+, activated the same 3-pS channels in
cell-attached membrane patches as well as whole cell nonselective
cation currents in SMC. TG- and BAPTA-activated 3-pS channels were
cation-selective but poorly discriminated among Ca2+,
Sr2+, Ba2+, Na+, K+,
and Cs+. Open channel probability did not change at
negative membrane potentials but increased significantly at high
positive potentials. Activation of 3-pS channels did not depend on
intracellular Ca2+ concentration. Neither TG nor a variety
of second messengers (including Ca2+, InsP3, InsP4,
GTP Depletion of intracellular Ca2+ stores is known to
activate store-operated (or capacitative) Ca2+ influx in a
variety of nonexcitable cells (for review, see Refs. 1-5). The idea of
capacitative Ca2+ entry
(CCE)1 was initially proposed
for smooth muscle cells (SMC) (6). Over the past years it has been
shown that CCE and contraction can be activated in SMC by passive
depletion of intracellular Ca2+ stores even without
activation of receptor-dependent cascades (for review, see
Ref. 7), although the nature of the ion channels responsible for CCE in
vascular SMC remains obscure. In some nonexcitable cells, highly
Ca2+-selective calcium release-activated calcium (CRAC)
channels (4, 8) and certain members of the diverse family of TRP
channels (9, 10) are thought to be responsible for CCE. However, to date the existence of neither of those has been established in SMC.
Importantly, depletion of Ca2+ stores was shown to trigger
not only Ca2+, but also Na+ influx in arterial
myocytes (11), which implies that store-operated channels in SMC are
poorly selective for cations. In freshly isolated mouse anococcygeus
SMC there are strong indications that CCE results from activation of a
whole cell nonselective cation current (12, 13), although in rat aortic
SMC line A7r5 no currents were detected which could be associated with
CCE (14, 15). It is totally unclear if the same or different
store-operated channels mediate CCE in SMC from different preparations.
Here for the first time we characterize 3-pS cation channels that are
activated by Ca2+ store depletion in intact SMC from mouse
and rabbit aorta. These channels, contrary to highly
Ca2+-selective CRAC channels, are poorly selective for
mono- and divalent cations, and under physiological conditions they
will allow both Ca2+ and Na+ to enter SMC.
Recently we found that these channels can also be activated in excised
membrane patches by calcium influx factor (CIF) partially purified from
human platelets or yeast with depleted Ca2+ stores (16).
Taken together, these data strongly support the idea that the native
3-pS channels, which we found in SMC, belong to the class of
store-operated ion channels. Preliminary data have been reported in
abstract form (17, 18).
SMC Preparation
Four different preparations of aortic SMC were used in our
experiments, and the 3-pS channel described in this paper was found to
be the same in acutely dispersed and cultured SMC from mouse and rabbit
aorta. Most of the experiments on characterization of the single
channels and whole cell currents were done on mSMC in short term
culture because they provided the most reliable model for studies of
single channels, whole cell currents, and intracellular
Ca2+.
Mouse SMC (mSMC)
Mouse SMC were isolated from thoracic aorta of C57BL6 mice
(15- 20 g). Two animals were anesthetized by inhalation of halothane and killed by cervical dislocation. The thoracic aortas were rapidly removed, cleaned of connective tissues, and cut into small pieces.
Acutely Dissociated mSMC--
Acutely dissociated mSMC were
obtained as described previously (19). Briefly, pieces of aorta were
incubated in dissociation medium (DM, in mM: 110 NaCl, 5 KCl, 10 NaHCO3, 0.5 NaH2PO4, 0.5 KH2PO4, 2 MgCl2, 10 taurine, 10 HEPES, 11 glucose, and 0.02% bovine serum albumin, pH 7.2)
supplemented with 40 units/ml papain and 2 mM
dithiothreitol for 15 min at 37 °C with constant stirring. After
incubation, pieces of aorta were rinsed twice in fresh DM and then
gently triturated with a heat-polished Pasteur pipette. Isolated cells
were stored at 4 °C until use for up to 4 h. For experiments,
20 µl of the cell suspension was placed in a 35-mm polystyrene tissue
culture dish or a 0.15-mm glass-bottom chamber, and SMC were allowed to
adhere to the bottom before the beginning of the experiment.
Mouse SMC in Short Term Primary Culture--
Mouse SMC cultures
were prepared in the following way. SMC were acutely dissociated by
incubation of pieces of mouse aorta in 2 ml of Dulbecco's modified
Eagle's medium containing 4 mg/ml collagenase, 1.5 mg/ml elastase, and
0.5 mg/ml trypsin inhibitor for 1 h at 37 °C. During
incubation, tissue was gently triturated every 15 min. Enzymatic
digestion was terminated by the addition of 5 ml of fresh Dulbecco's
modified Eagle's medium (free of enzymes) supplemented with 10% fetal
bovine serum (FBS). The cell suspension was centrifuged, supernatant
discarded, and the pellet was resuspended in 2 ml of Dulbecco's
modified Eagle's medium containing 1% fetal bovine serum, 100 units/ml penicillin G, and 10 mg/ml streptomycin. The cell suspension
was placed on coverslips and kept in 35-mm Petri dishes at 37 °C in
5% CO2 for 3-7 days. Under these conditions, mSMC
attached to the coverslips but did not divide, had a bipolar morphology, and stained positively for Rabbit Aortic SMC (rSMC)
Male New Zealand White rabbits (2-2.5 kg) were exsanguinated
after injection of 30 mg/kg sodium pentobarbital and 150 units/kg heparin. A segment of thoracic aorta was rapidly removed, cleaned of
connective tissues, cut into small pieces, and rinsed in DM.
Acutely Dissociated rSMC--
Acutely dissociated rSMC were
obtained by incubation of the pieces of rabbit aorta in DM with 50 units/ml papain and 2 mM dithiothreitol for 30 min at
37 °C in a shaking water bath as described previously (19). After
incubation, they were rinsed twice in fresh DM and then gently
triturated with a heat-polished Pasteur pipette. Isolated cells were
stored at 4 °C until use for up to 4 h.
Primary Cultured rSMC--
Rabbit SMC cultures were prepared as
described previously (20). Experiments on mice and rabbits were
conducted in accordance with the Guide for the Care and Use of
Laboratory Animals (57).
Tension Measurements
Isometric tension was measured in the rings of mouse thoracic
aorta as described previously (20).
Ca2+ Measurements
Cells were incubated with 2.5 µM fura-2/AM + 0.02% pluronic acid for 30 min at room temperature and subsequently
washed for 15 min. Fluorescence (excitation at 340 and 380 nm, emission
at 510 nm) was measured at 20-22 °C using an inverted microscope (Olympus IX70, Japan) equipped with a 20 × fluorescence objective and dual excitation fluorescence imaging system (IonOptix Inc.). Changes in intracellular Ca2+ concentration in mSMC were
determined using standard methods as described earlier (19).
Electrophysiology
Single channel and whole cell currents were recorded as
described recently (19) with a low noise Axopatch 200B amplifier (Axon
Instruments). pCLAMP 6 software (Axon Instruments) was used for data
acquisition and analysis. Data were filtered at 1 kHz and stored for
later analysis. Representative traces of single channel currents were
later filtered additionally at 100-200 Hz for better visual resolution
of 3-pS single channels on the figures. Experiments were conducted at
20-22 °C.
Single Channel Currents--
Currents were recorded in the
cell-attached and inside-out membrane patches. To improve the
signal-to-noise ratio, pipettes were coated with Sylgard (Dow Corning
Corp.) and polished to a resistance of 10-20 megohms (when filled with
high Na+ pipette solution). Single channel currents were
recorded at ±100, ±80, and ± 60 mV applied with respect to the
cytosolic side of membrane. Inward and outward currents are shown as
downward and upward deflections, respectively, from the base line
(labeled 0 on the figures). The amplitude of single channel
currents was analyzed using all point histograms (see Fig.
2E) or amplitude histograms obtained from the event list
(see Fig. 2C). Both methods gave the same single channel
current amplitude for the first two current levels (Fig. 2,
C and E), but we found the second method to be
better for resolving infrequent channel openings in resting SMC (see
Fig. 2B) or when more than two single channel current levels
were observed at the peak of channel activity (see Fig. 2C).
The open channel probability (NPo) was analyzed and plotted over time to illustrate the time course of channel activity. The total
apparent number of channels (N) in individual patches was estimated after their activation and was based on the maximum number of
single channel current levels observed simultaneously at +100 mV. In
each membrane patch n varied from 3 to 5 (average N = 4). Because prolonged full activation of all of the
channels has not been achieved in some experiments, we cannot exclude
the possibility that the actual number of the channels in each patch could be slightly higher than the apparent one. Standard bath solution
contained (in mM) 140 NaCl, 2.8 KCl, 2 MgCl2,
5.5 glucose, 10 HEPES (pH 7.4). Standard pipette solution contained (in
mM) 140 NaCl, 10 TEA, 0.2 EGTA, 10 HEPES (pH 7.4).
In some experiments NaCl in the pipettes was replaced by KCl, CsCl,
CaCl2, BaCl2, SrCl2, sodium
glutamate, or NMDG-Cl (as specified in Table I). The
liquid junction potential was compensated. In some experiments Ca2+ (1 or 10 mM) was added to 140 mM NaCl-containing pipette or bath solutions. The pipette
solutions also contained 100 µM niflumic acid and 100 nM iberiotoxin to prevent activation of
Cl
Because of the extremely small amplitude of single channel currents we
were able to record and analyze them only at membrane potentials
between ±60 and ±100 mV. For that reason it was impossible to
determine experimentally and compare the exact reversal potential of
single channel currents under different ion conditions and to provide
the standard calculations of channel selectivity for different cations
based on the shifts of the reversal potential of single channel
current. Because of these technical limitations we only determined the
relative cation conductivity of single channels under different ionic
conditions (Table I), which was estimated
from the slope conductance of inward or outward current in the presence
of different cations. The slope conductance was calculated from the
single channel currents at a minimum of three different membrane
potentials.
Whole Cell Currents--
Currents were recorded using
conventional whole cell configuration. Bath solution contained (in
mM) 130 NaCl, 2.8 KCl, 1 MgCl2, 10 HEPES, 10 TEA, 100 µM niflumic acid (pH 7.4), and 0.1 µM Ca2+ (buffered with EGTA). The pipette
solution contained (in mM) 60 aspartic acid, 40 CsCl, 4.5 NaCl, 10 HEPES, and 10 BAPTA-Cs4 (pH 7.2 with CsOH).
Pipettes had resistance of 2-5 megohms when filled with pipette
solution. Development of the whole cell inward current was monitored at
holding potential of Drugs
Acetoxymethyl ester of BAPTA (BAPTA/AM) and fura 2-AM were from
Molecular Probes Inc. (Eugene, OR). Papain was from Fluka (Bucks,
Switzerland). All other drugs were from Sigma.
Statistics
The data are presented as means ± S.E. with n
showing the number of experiments. Statistical significance was
assessed using the t test and analysis of variance. Values
of p < 0.05 were considered significant.
Thapsigargin-induced Contraction and Intracellular Ca2+
Rise in Aortic SMC--
5 µM TG applied to the intact
mouse aorta (Fig. 1A) caused a
substantial contraction (610 ± 22 mg), but only after the
addition of 2 mM extracellular Ca2+, which is
consistent with TG-induced activation of Ca2+ influx. In
individual SMC, TG applied in the absence of extracellular Ca2+ (Fig. 1B) caused a small increase in
[Ca2+]i which can be explained by
passive Ca2+ release from intracellular stores leading to
their depletion. Subsequent addition of 5 mM
Ca2+ caused a sustained rise in
[Ca2+]i, which reflects TG-induced
activation of Ca2+ influx (summary data are shown in Fig.
1C). Both Ca2+ influx-dependent
[Ca2+]i rise and contraction were
inhibited by 2-5 mM nickel, (Fig. 1).
Small Conductance Channels Activated by Thapsigargin in Aortic
SMC--
To define the nature of the ion channels that are responsible
for TG-induced Ca2+ influx, single channel currents were
recorded in cell-attached membrane patches in mouse (mSMC) and rabbit
(rSMC) aortic SMC. Extracellular application of 2 µM TG
activated small conductance channels in cell-attached membrane patches
in mSMC and rSMC which were acutely dispersed or cultured. Fig.
2 shows a typical example (n = 31 out of 52) of single channel activity and
current amplitude in cell-attached membrane patches in mSMC (short term
culture) before and after TG application with original traces of single channel outward currents (measured at +100 mV applied with respect to
the inside of membrane) at different times of the experiment. Only rare
single channel openings could be detected in the resting cells.
However, 40-180 s after the application of TG, three to five single
channel current levels were observed. Although the amplitude of single
channel currents activated by TG was very small, it could be analyzed
using either all point histograms (Fig. 2E) or event list
histograms (Fig. 2C), which gave similar values of single
channel current amplitude.
TG-induced activation of single channel currents was observed in
control mSMC (n = 10) and in mSMC with intracellular
Ca2+ buffered with 20 µM BAPTA/AM for 20 min,
(n = 21). Importantly, in about 30% of SMC, BAPTA
loading itself caused activation of the same channels as will be
described below. TG-activated small single channel currents were
observed in the absence or presence of 100 nM iberiotoxin
and 100 µM niflumic acid, inhibitors of K
It is important to emphasize that the same small conductance channels
were activated by TG in acutely dispersed mSMC and rSMC as well as in
rSMC in primary culture. As an example, Fig.
3A shows TG-activated inward single
channel currents and their amplitude histogram in a cell-attached
membrane patch in SMC acutely dispersed from rabbit aorta
(n = 5), which were indistinguishable from those found
in mSMC (Fig. 2). Detailed characteristics of TG-activated channels in
mSMC and rSMC are presented below.
Main Characteristics of TG-activated Channels in Inside-out
Membrane Patches--
After TG-induced activation in cell-attached
patches, single channels remained active for 5-15 min even when the
membrane patches were excised from SMC in Ca2+-free
solution (Fig. 2D, n = 4 out of 4). 2 mM LaCl3 applied to the inside of membrane
patches inhibited single channel currents (n = 4).
Importantly, excision of membrane patches from resting mSMC (not
treated with TG or BAPTA/AM) in the presence or absence of 1 µM or 1 mM CaCl2 did not by
itself activate the channels (n = 54).
Single channel currents in inside-out membrane patches from
TG-activated mSMC and rSMC (acutely dispersed or cultured) had identical properties, and Fig. 4 shows a
typical example of single channel currents recorded in inside-out
membrane patches with amplitude histograms at different membrane
potentials. Current-voltage (I/V) relationships of single channels
under different ionic conditions in inside-out membrane patches from
rSMC and mSMC are shown in Fig. 3, B and C,
respectively (acutely dissociated cells) and Fig.
5, A and B
(cultured cells). The slope conductance in symmetrical 140 mM NaCl was 3.3 ± 0.1 pS (n = 4) in
acutely dissociated and 3.4 ± 0.2 pS (n = 6) in
cultured mSMC, which was similar to acutely dissociated and cultured
rSMC (3.3 ± 0.1 pS, n = 8 and 3.2 ± 0.1 pS,
n = 9, respectively). When Na+ in the
pipette was replaced by 100 mM NMDG-Cl, inward single channel currents (at
In inside-out membrane patches, the NPo of single channels
in both mSMC (Fig. 5C) and rSMC (Fig. 5D) was
similar and did not change much at negative membrane potentials
(NPo
Thus, the same channels were activated by TG in acutely dissociated or
cultured SMC from mouse and rabbit aorta, which appeared to be
nonselective cation channels with about a 3-pS conductance.
Importantly, application of 2-5 µM TG (n = 12), 1 µM Ca2+ (n = 12) or
1 mM Ca2+ (n = 42), 20 µM InsP3 (n = 11), 10 µM InsP4 (n =5), 200 µM GTP Loading SMC with BAPTA Activated 3-pS Nonselective Cation Channels
and Whole Cell Currents--
BAPTA, a high affinity Ca2+
chelator, activates CRAC currents in a variety of nonexcitable cells
and is commonly used to deplete intracellular Ca2+ stores
without an increase in [Ca2+]i or
activation of receptor-dependent pathways (for review, see
Refs. 4 and 21). Dialysis of mSMC with 10 mM BAPTA in the
absence of extracellular Ca2+ slowly activated an inward
current (at
When mSMC were loaded with 20 µM BAPTA/AM for 20 min,
small conductance channels were activated in 25 out of 72 cells, which were the same as 3-pS channels activated by TG. Fig. 6F
shows an example of single channel currents and their amplitude
histograms at different membrane potentials in a membrane patch excised
from a BAPTA-loaded mSMC. The I/V relationship (Fig. 6E) and
the increase in NPo at high positive membrane potentials
(Fig. 6D) of BAPTA-activated channels were similar to the
3-pS channels activated by TG in mSMC and rSMC (see Fig. 5). In mSMC
not loaded with BAPTA, spontaneous activation of 3-pS channels was
observed only in 3 out of 26 cells.
Because BAPTA-activated 3-pS channels and whole cell currents were both
poorly cation-selective and both showed significant outward
rectification at high positive membrane potentials, we determined
whether activation of 3-pS channels could indeed produce the whole cell
current with characteristics of that experimentally observed in
BAPTA-loaded mSMC. The amplitudes of the hypothetical whole cell
current were calculated from the I/V relationship (Fig. 6D)
and NPo (Fig. 6E) of single channel currents
(assuming that about 1,000 membrane patches could comprise about a
2,000-µm2 surface of mSMC). As predicted, the calculated
current amplitudes (Fig. 6C, open circles) were
close to the amplitude of the actual whole cell current (Fig.
6C, closed diamonds) recorded in mSMC loaded with
BAPTA. This result strongly suggests that 3-pS nonselective cation
channels are responsible for the whole cell current activated by BAPTA
in mSMC.
The present study provides the first insight into the nature of
single ion channels that are responsible for store-operated Ca2+ influx in vascular SMC. We found
Ca2+-conducting nonselective cation channels of very small
(3-pS) conductance which are activated after loading mSMC with BAPTA and/or treatment with TG, which are commonly used to passively deplete
intracellular Ca2+ stores. The very small amplitude of
single channel currents activated by TG and BAPTA in mouse and rabbit
aortic SMC might explain why these channels have not been described
previously. Indeed, many different types of nonselective cation
channels have been described in SMC (15, 22-37), but the single
channel properties and mechanism of activation of the 3-pS channel
distinguish it from all of the channels found in SMC so far (for
review, see Refs. 5, 38, and 39). Our results strongly support the
possibility that novel 3-pS channels are store-operated and are
responsible for TG-induced Ca2+ influx in individual SMC
and contraction of the aorta.
Characteristics of the 3-pS Channel in SMC--
Even though the
amplitude of single channel currents did not exceed 0.4 pA even at ± 100 mV, the low noise patch-clamp system allowed us to resolve
clearly single channel openings and to perform some analysis of their
amplitude and open channel probability. TG- and BAPTA-activated
channels appeared to be nonselective cation channels that conducted and
poorly discriminated between monovalent (Na+,
K+, Cs+) and divalent (Ca2+,
Sr2+, Ba2+) cations. Their single channel
conductance was close to 3 pS for a variety of cations tested. As a
result of activation of such nonselective cation channels both
Ca2+ and Na+ are expected to enter SMC causing
not only a rise in intracellular Ca2+, but also significant
influx of Na+. Indeed, depletion of Ca2+ stores
has recently been shown to activate both Ca2+ and
Na+ influx in arterial myocytes (11). Influx of
Na+ into SMC could result in membrane depolarization that
could potentially trigger activation of voltage-dependent
Ca2+-selective channels that might contribute to additional
Ca2+ influx and SMC contraction. Single channel recording
during SMC activation with TG provides some indirect evidence of
TG-induced SMC depolarization. Indeed, rare openings of single channels
in resting SMC result in outward currents of about 0.22 pA at +100 mV
applied in respect to the cytoplasmic side of cell-attached membrane
patches (Fig. 2, A and B). Simple calculations
show that this amplitude is about what one would expect from a 3.4-pS
channel if the resting membrane potential is around 3-pS Nonselective Cation Channels Could Underlie BAPTA-activated
Whole Cell Current--
A critical "trademark" feature of
store-operated channels is that they can be activated independently of
intracellular Ca2+ rise and major signaling cascades. Such
conditions are achieved by cell dialysis with 10-20 mM
BAPTA. Indeed, BAPTA provides a fast and strong buffering of
intracellular Ca2+, preventing Ca2+ stores from
refilling and promoting their passive depletion. Extracellular
Ca2+ must also be eliminated to prevent saturation of BAPTA
by Ca2+ entering the cell. Activation of the whole cell
current under these conditions is thought to be one of the strongest
pieces of evidence for the existence of store-operated channels in
different types of cells (4).
Dialysis of mSMC with 10 mM BAPTA in our experiments
activated a poorly selective whole cell current with a reversal
potential around 0 mV (please note that Cl
Thus, the BAPTA-activated current is likely to result from activation
of the 3-pS nonselective cation channels found in the same mSMC. We
estimated that about 5,000 channels must be present in mSMC to account
for the BAPTA-activated whole cell current. Indeed, at 50 mV the
average whole cell inward current (I) of about 40 pA
develops after 3 min in BAPTA-loaded mSMC (Fig. 6B). Assuming that the single channel current at Native 3-pS Channels Are Not Regulated by Intracellular
Ca2+--
Application of TG to intact SMC causes a
pronounced increase in [Ca2+]i,
and some of the channels could be activated by TG-induced intracellular
Ca2+ rise rather than by depletion of Ca2+
stores. Several lines of evidence obtained in our experiments exclude
this possibility. First, 3-pS channels were not activated directly by
Ca2+ when inside-out membrane patches were excised into
Ca2+-containing solutions. Two different concentrations of
Ca2+ in the bath were used in these experiments (1 µM and 1 mM) to ensure that the possibility
of a bell-shaped dependence on Ca2+ concentration was not
overlooked. For comparison, in our recent studies (19), we demonstrated
that excision of membrane patches from the same cells into
Ca2+-containing solution activated
Ca2+-dependent Cl 3-pS Nonselective Cation Channels Are Likely to Be Native
Store-operated Channels in SMC--
The results of our studies provide
several lines of evidence that in intact SMC 3-pS channels are
activated by depletion of intracellular Ca2+ stores rather
than by other mechanisms. First, we found 3-pS channels to be activated
by TG and BAPTA, which are known to cause passive depletion of
Ca2+ stores and activation of store-operated channels and
whole cell currents in a variety of nonexcitable cells. Second,
activation of 3-pS channels and corresponding whole cell currents do
not require intracellular Ca2+ rise or activation of
InsP3, or other receptor-dependent
cascades and could be achieved by simple loading of SMC with the
Ca2+ chelator BAPTA. Third, a variety of second messengers
that are known to be involved in Ca2+ homeostasis of SMC
(Ca2+, InsP3, InsP4, GTP
Recently, we found that 3-pS nonselective cation channels in inside-out
membrane patches from SMC are activated by CIF partially purified or
bioassayed from yeast or human platelets with depleted Ca2+
stores (16). This CIF is thought to be one of the possible messengers
produced by endoplasmic reticulum upon depletion of Ca2+
stores which can activate plasma membrane channels and cause CCE. These
data strongly support the idea that 3-pS nonselective cation channels
(which we showed here to be activated in intact SMC upon depletion of
their stores) indeed belong to the class of store-operated channels.
Although their electrophysiological characteristics are significantly
different, activation properties of 3-pS cation channels and whole cell
currents resemble those of other store-operated channels and currents
in nonexcitable cells. The onset of TG-induced activation of 3-pS
channels falls within the time frame of passive depletion of
Ca2+ stores when sarco-endoplasmic
reticulum-dependent back-sequestration of Ca2+
is inhibited. After being activated, 3-pS channels remain active for
several minutes even after inside-out patches are excised from SMC,
which is similar to the store-operated currents recorded in giant
inside-out membrane patches from Xenopus oocytes (41). BAPTA
loading of SMC also activates the whole cell nonselective cation
current with a time course similar to that of activation of
ICRAC. However, the electrophysiological characteristics of the currents in SMC are different from that known for CRAC channels (for review, see Ref. 4). For example, ICRAC in
nonexcitable cells is highly Ca2+-selective and conducts
Na+ only in the absence of Ca2+, whereas BAPTA
and TG-activated single channels in SMC are poorly cation-selective,
and their Na+ conductance does not change significantly in
the presence or absence of Ca2+. Also, in the presence of
Ca2+ on one side of the membrane and Na+ on the
other, the I/V relationship of 3-pS channels is linear with
approximated reversal potential around zero mV. In the presence of
Na+ and Ca2+ the whole cell current in SMC
reverses at 0 mV and has significant outward rectification, whereas
ICRAC reverses at high positive potentials (around the
Ca2+ equilibrium potential) and has a very pronounced
inward rectification. The whole cell currents and single 3-pS channels
could be inhibited by millimolar concentrations of nickel or lanthanum
(above 2 mM), whereas ICRAC is inhibited by
lanthanum in the micromolar range. Finally, under the same ionic
conditions (in the presence of Na+ and Ca2+)
the conductance of CRAC channels was estimated to be about 9 fS (42),
which is 300 times less than the 3-pS conductance we observed. Thus,
selectivity and conductance of 3-pS channels in SMC are clearly
different from CRAC channels found in nonexcitable cells, but that does
not exclude the possibility that both channels could be similarly
regulated by the filling state of intracellular Ca2+ stores.
The poor cation selectivity of TG- and BAPTA-activated channels in SMC
resembles that of the TRP1 channel expressed in some mammalian
cells (43-45), although its conductance was estimated to be around 16 pS. The possibility that 3-pS nonselective cation channels in SMC could
be related to the growing family of TRP channels needs further investigation.
Physiological Relevance of Store-operated Cation Channels in
Vascular SMC--
The existence of store-operated Ca2+
influx in vascular SMC is strongly supported by many studies that
demonstrated Ca2+ influx in SMC and contractions in
different vessels caused by sarco-endoplasmic reticulum
Ca2+ ATPase inhibitors (12, 15, 35, 46-56). Recently, we
demonstrated that this pathway in vascular SMC is regulated by nitric
oxide, the major endothelium-derived relaxing factor, which indirectly inhibits CCE by enhancing sarco-endoplasmic reticulum
Ca2+ ATPase-dependent refilling
of Ca2+ stores (20). This implies the physiological
importance of CCE in regulation of vascular tone, but the nature of the
channels involved in this store-regulated process remains obscure.
Indeed, studies in a fetal rat aortic cell line (A7r5) failed to reveal single channels or cation currents activated by TG (14, 15), although
in freshly isolated mouse anococcygeus SMC a whole cell nonselective
cation current was described which correlated with capacitative
Ca2+ influx (12, 13).
Our studies for the first time demonstrate the presence of small
conductance (3-pS) nonselective cation channels in intact SMC from
mouse and rabbit aorta which are activated by depletion of
intracellular Ca2+ stores (with BAPTA or TG). Importantly,
we found and described these channels in both acutely dispersed and
cultured SMC. These new data, together with demonstration of TG-induced
Ca2+ influx in isolated mSMC (Fig. 1), TG-induced
Ca2+ influx in intact smooth muscle strips of mouse aorta
(20), and TG-induced contraction of the mouse aorta (Fig. 1),
strongly suggest that TG-activated 3-pS cation channels could be
responsible for store-operated Ca2+ influx and contraction
of aortic SMC.
At the end, it is important to mention that although we found 3-pS
channels in both freshly dispersed and cultured SMC, it was easier to
record and study them in cultured SMC. Also, SMC in culture showed more
consistent responses to TG (judged by TG-induced Ca2+
influx), which was observed in 80-90% of cells, whereas such responses in acutely dissociated cells varied from isolation to isolation, and on average TG-induced Ca2+ influx was
observed in only about 10% of freshly dissociated SMC. Such
differences in the number of cells responding to TG most probably
reflect the result of enzymatic shock which is unavoidable during the
acute isolation of fresh SMC but can be reduced if SMC are allowed to
recover for a few days in short term culture. Culture of the cells does
not apparently change the electrophysiological properties of these 3-pS
channels. This simple explanation is also supported by the fact that
TG-induced Ca2+ influx is present in intact smooth muscle
strips of mouse aorta (20) before their exposure to enzymes.
Interestingly, the same methods of acute isolation of SMC from mouse
and rabbit aorta did not affect SMC responsiveness to caffeine which
causes Ca2+ release from the stores and activation of
Ca2+-dependent ClS, cyclic AMP, cyclic GMP, ATP, and ADP) activated 3-pS channels
in inside-out membrane patches. Thus, 3-pS nonselective cation channels
are present and activated by TG or BAPTA-induced depletion of
intracellular Ca2+ stores in intact SMC. These native
store-operated cation channels can account for capacitative
Ca2+ influx in SMC and can play an important role in
regulation of vascular tone.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin.
Single channel conductance under different ionic conditions
50 mV with voltage ramp (from
100 to +50 mV,
500 ms) applied every 2 s. Because whole cell currents showed a
strong outward rectification at high positive potentials and because
SMC loaded with BAPTA (in the absence of extracellular
Ca2+) were very fragile, whole cell current recordings at
potentials higher than +50 mV were generally avoided in our
experiments. Leak current was not subtracted.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (16K):
[in a new window]
Fig. 1.
TG-induced contraction of the whole mouse
aorta (panel A) and changes in intracellular
Ca2+ in single isolated mSMC
(panels B and
C). Panel A, isometric tension
in the ring of mouse thoracic aorta during the application of 4 µM TG in the absence of extracellular Ca2+
and after 2.5 mM Ca2+ and 2 mM
Ni2+ were added. Panel B, representative
recording of [Ca2+]i in mSMC
(short term culture). 2 µM TG was applied (open
horizontal bar above the trace) in the absence of
extracellular Ca2+ followed by 2 mM
Ca2+ (closed bar) and 4 mM
Ni2+ (hatched bar). Panel C, summary
data from 16 experiments similar to that shown in panel B.
The bars represent Ca2+ levels before
(1) and after the addition of TG (2),
Ca2+ (3), and Ni2+
(4).
View larger version (28K):
[in a new window]
Fig. 2.
TG-activated single channel currents in
cultured mSMC. Panel A, NPo in the
cell-attached membrane patch before and after the application of 2 µM TG (open bar) to intact mSMC (in short term
culture) loaded with 20 µM BAPTA/AM for 20 min. A similar
TG-induced activation of small conductance channels was observed in 31 out of 52 cells. Below, the original traces of single channel outward
currents at +100 mV applied to the membrane (equivalent to 100 mV
applied to the pipette) are shown at different times of the experiment.
The closed state of the channel is marked by 0, and open
states are labeled as 1 and 2 at the beginning of
the original traces. Panels B and C, amplitude
histograms (from the event list) of a single channel current before
(panel B) and after (panel C) the application of
TG (from the experiment shown in panel A). The
numbers above each peak represent the amplitude of
corresponding current level. Panel D, example of single
channel currents recorded in a cell-attached membrane patch in SMC
pretreated with TG (TG) and after the inside-out patch was
excised (I/O) into Ca2+-free solution containing
5 mM EGTA (0 Cai). Panel
E, all-point amplitude histogram from the same single channel
current recording as in panel C. The amplitudes of different
current levels are shown above each peak.
View larger version (19K):
[in a new window]
Fig. 3.
TG-activated single channel currents in fresh
SMC. Panel A, original traces of single channel inward
currents recorded cell-attached membrane patch (at 100 mV) before
(control) and 4 min after the application of 2 µM
TG to intact acutely dissociated rabbit SMC. The closed and open
states of the channel are marked by 0 and 1,
respectively (openings are downward deflections). All point
amplitude histograms of single channel current after the application of
TG are shown on the right. Panel B,
current-voltage relationship of TG-activated single channels in
inside-out membrane patches from acutely dissociated rabbit SMC in
symmetrical 140 mM NaCl. Summary data are from eight
experiments with S.E. bars shown where they exceed the size of the
symbol. Panel C, same as in panel B
but for TG-activated channels from acutely dissociated mSMC. 140 mM CsCl was in the pipette and 140 mM NaCl in
the bath. Summary data are from four experiments with S.E. bars shown
where they exceed the size of the symbol.
100 mV) disappeared, although outward currents recorded at +100 mV in the same membrane patches did not change (n = 3, Fig. 5A). Replacement of
Cl
with glutamate in the pipette did not affect single
channel currents at both positive and negative membrane potentials
(3.4 ± 0.6 pS, n = 3, Fig. 5A),
confirming that the 3-pS channel is cation-selective. Fig.
5A shows that the conductance of 3-pS channels did not
change significantly when Ca2+ was added to
Na+-containing pipette solution at concentration of 1 mM (3.3 ± 0.7 pS, n = 16) or 10 mM Ca2+ (2.9 ± 0.8 pS, n = 3), which showed that, contrary to Ca2+- selective
channels, the 3-pS channel does not prefer Ca2+ over
Na+ when both cations are present. When Na+ in
the pipette was replaced by K+ or Cs+, the
single channel inward current had similar slope conductance of 3.4 ± 0.1 pS (n = 3) and 3.4 ± 0.2 pS
(n = 4), respectively, which was similar to that in
Na+. To test if 3-pS channels along with monovalent cations
also conduct divalent cations, Na+ in the pipette and/or in
the bath was replaced by Ca2+, Sr2+, or
Ba2+ (Fig. 5, A and B). When 90 mM Ca2+ was used in the pipette and 90 mM Ba2+ was used in the bath (with no
Na+ present on either side), both inward and outward
currents were observed, and the slope conductance of inward
(Ca2+) current was 2.7 ± 0.1 pS, and the outward
(Ba2+) current was 3.5 ± 0.1 pS (Fig. 5B,
n = 8). When 90 mM Ca2+ or
Sr2+ was in the pipette and 140 mM
Na+ was in the bath, the slope conductance of inward
current was 3.0 ± 0.6 pS (for Ca2+, n = 3) and 2.7 ± 0.1 pS (for Sr2+, n = 9), with no apparent change in slope conductance of outward (Na+) current. Working at the limits of resolution of
single channel currents, we were not able to determine the exact
reversal potential of single channel current in mixed solutions which
is required for estimation of exact channel selectivity to different
cations. Minor changes in conductivity of the channel in the presence
of different cations (summarized in Table I) would suggest that these
TG-activated 3-pS channels are cation-selective but discriminate poorly
between different mono- and divalent cations, and thus under
physiological conditions would allow both Ca2+ and
Na+ to enter SMC.
View larger version (29K):
[in a new window]
Fig. 4.
TG-activated single channel currents in
inside-out membrane patches at different potentials. This is a
typical example of TG-activated single channel currents recorded in
inside-out membrane patch (under symmetrical 140 mM NaCl
conditions) at the indicated potentials applied with respect to the
inside of the membrane. Single channel openings are shown as
upward (at positive membrane potentials) or
downward (at negative membrane potentials) deflections. The
closed state of the channel is shown by 0; open states are
labeled 1, 2, and 3 at the beginning
of the original traces. The panels on the right
represent all points histograms calculated for each trace. The membrane
patch is from cultured rabbit SMC.
View larger version (17K):
[in a new window]
Fig. 5.
Properties of single channels activated by TG
in mouse (panels A and C) and rabbit
(panels B and D) SMC. Panel
A, current-voltage relationship of TG-activated single channels in
inside-out membrane patches from cultured mSMC. The ion composition of
the pipette solution was (in mM) 140 NaCl ( ), 140 NaCl
and 1 CaCl2 (
), 140 NaCl and 10 CaCl2 (
),
90 CaCl2 (
), 100 NMDG (
) or 100 sodium glutamate
(
) with 10 HEPES (pH 7.4). In all experiments the ion composition of
the bath solution was (in mM) 140 NaCl, 1 Ca2+,
2 Mg2+, 2.8 KCl, and 10 HEPES. Each point is an average of
3-16 experiments with S.E. bars shown where they exceed the size of
the symbol. Panel B, same as in panel
A but for single TG-activated channels from cultured rabbit SMC.
The ion composition of the pipette solution was (in mM) 140 NaCl (
), 90 CaCl2 (
), 90 SrCl2 (
), or
140 KCl (
). The bath contained (in mM) 140 NaCl (
),
90 BaCl2 (
), or 90 SrCl2 (
). 10 mM HEPES (pH 7.4) was present in all solutions. Panel
C, voltage dependence of the NPo in inside-out
membrane patches from TG-activated cultured mSMC. Summary data are from
18 experiments. Panel D, same as in panel C but
for channels from TG-activated cultured rabbit SMC. Summary data are
from 6 experiments.
0.2 at-60 mV) but significantly increased at high
positive membrane potentials. Although only one level of single channel
currents was usually seen at
100 mV, activation of three to five
levels (average four, n = 28) could be detected at +100
mV (Fig. 4). Thus, in the physiological range of membrane potentials
the Po of each individual channel activated by TG in SMC
could be as low as 0.05.
S (n = 5), 100 µM
cyclic AMP (n = 5), 100 µM cyclic GMP
(n = 5), 1.7 mM ATP (n = 6), or 100 µM ADP (n = 5) to inside-out
membrane patches excised from the resting mSMC did not activate 3-pS channels.
50 mV) which usually started after 40-60 s and reached a
stable plateau of 39 ± 13 pA (n = 6) within 2-3
min (Fig. 6, A and
B). The current was inhibited by 2 mM
LaCl3, n = 5). The I/V relationship of the
BAPTA-induced whole cell current (Fig. 6C) reversed at
2.5 ± 1.4 mV and showed strong outward rectification, especially
at high positive potentials (not shown).
View larger version (27K):
[in a new window]
Fig. 6.
Single channels and whole cell currents
activated by SMC loading with BAPTA. Panel A,
representative experiment showing the changes in the amplitude of the
whole cell current (at 50 mV) during the dialysis of mSMC with 10 mM BAPTA (open bar) and after the application of
2 mM La3+ (closed bar). Panel
B, summary data showing the amplitudes of the whole cell currents
in mSMC at the moment of breaking into the cell (0"),
after 60 and 180 s of SMC dialysis with 10 mM BAPTA,
and after application of lanthanum (+La) in six experiments
similar to the one shown in panel A. Panel C,
summary of six experiments showing the current-voltage relationship
(
) of the peak whole cell current activated in SMC by cell dialysis
with 10 mM BAPTA.
, estimated amplitude of the
hypothetical whole cell current calculated from the I/V relationship
(panel E) and NPo (panel D) of 3-pS
single channel currents (for details, see "Results").
Panel D, voltage dependence of NPo of 3-pS
channels in inside-out membrane patches from mSMC activated by loading
with 20 µM BAPTA/AM for 20 min. Panel E,
summary of 25 experiments showing current voltage (I/V) relationship of
single channels in inside-out membrane patches (under symmetrical 140 mM NaCl conditions) from mSMC activated by loading with
BAPTA/AM. Panel F, representative traces of single channel
currents at different membrane potentials in an inside-out membrane
patch from mSMC loaded with BAPTA/AM under symmetrical 140 mM NaCl conditions. Single channel openings are shown as
upward (at positive membrane potentials) or
downward (at negative membrane potentials) deflections. The
closed state of the channel is shown by 0; open states are
labeled 1, 2, and 3 at the beginning
of the original traces. Panels on the right
represent all points histograms calculated for each trace.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
40 mV (indeed we found the resting membrane potential of BAPTA-loaded SMC to be
39 ± 2 mV).2 After
robust activation of the channels by TG, their current amplitude
increased to about 0.31 pA (Fig. 2, A and C)
which is what could be expected from a 3.4-pS channel if the membrane
potential of SMC at that point is about
10 mV. Thus, the increase in
the amplitude of single channel outward currents in cell-attached membrane patches is consistent with significant TG-induced
depolarization of SMC. Importantly, 3-pS channels were found to be
present and open rarely in resting SMC. The activation of the channels
was observed within 1-3 min after the application of TG (Fig. 2), which is within the timeframe of the TG-induced onset of
Ca2+ influx in SMC and other cells (20, 40). The
NPo did not change significantly within the physiological
range of membrane potentials, but it increased dramatically at high
positive potentials (Fig. 5, C and D). This
increase of NPo was very typical for 3-pS channels in both
cell-attached and inside-out membrane patches, but the mechanism
underlying this phenomenon is unclear.
equilibrium
potential under our experimental conditions was
31 mV). The time
course of the development of this current was similar to that observed
for ICRAC in nonexcitable cells, but unlike
ICRAC, the current showed strong outward, rather than
inward rectification, under physiological ionic conditions (when both
Ca2+ and Na+ were present in the bath). Poor
cation selectivity and pronounced outward rectification strongly
resemble that of the 3-pS channels, which were also activated in mSMC
as a result of BAPTA/AM loading (Fig. 6). Moreover, the I/V
relationship of the inward and outward whole cell currents simulated
from single 3-pS channel currents (using their NPo and I/V
relationship) is similar to the BAPTA-activated whole cell current
recorded in mSMC.
50 mV is
= 0.16 pA, and each channel is opened with Po = 0.05, the minimum
number of the channels which could produce the whole cell current of 40 pA will be n = I/
Po = 5,000. If the channels are homogeneously distributed in the plasma membrane,
and an average mSMC has a capacitance of 20 picofarads and surface of
about 2,000 µm2, then two to three channels are expected
to be present in every 1 µm2 of plasma membrane. This is
in close agreement with an average of four channels which we observed
in 1-2-µm2 membrane patches. Thus, 3-pS channels are
likely to be responsible for the whole cell current activated during
mSMC cell dialysis with BAPTA.
channels (in
the absence of niflumic acid). These results show that contrary to
Cl
S,
cAMP, cGMP, ATP, ADP) did not activate single 3-pS channels in
inside-out membrane patches, excluding their role as physiological
activators of these channels.
channels (19)
in 90% of freshly dispersed cells. The reasons for the relative
partial impairment of capacitative Ca2+ influx mechanism
immediately after enzymatic treatment of SMC need further investigation.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. I. Medina for critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by NIH (HL54150, HL07224, and HL55993).
To whom correspondence should be addressed: Vascular Biology Unit,
Dept. of Medicine, Boston University School of Medicine, 650 Albany
St., X-704, Boston, MA 02118. Tel.: 617-638-7118; Fax: 617-638-7113; E-mail: vbolotina@med-med1.bu.edu.
Published, JBC Papers in Press, December 11, 2000, DOI 10.1074/jbc.M010104200
2 V. M. Bolotina and S. I. Zakharov, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: CCE, capacitative calcium entry; SMC, smooth muscle cell(s); CRAC, calcium release-activated calcium; mSMC, mouse aortic SMC; DM, dissociation medium; rSMC, rabbit aortic SMC; NPo, open channel probability; TG, thapsigargin; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; [Ca2+]i, intracellular Ca2+ concentration. TRP, transient receptor potential; CIF, calcium influx factor; NMDG, N-methyl-D-glucamine; InsP3, inositol trisphosphate; InsP4, inositol tetrakisphosphate.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Putney, J. W., Jr. (1986) Cell Calcium 7, 1-12[Medline] [Order article via Infotrieve] |
2. | Berridge, M. J. (1995) Biochem. J. 312, 1-11[Medline] [Order article via Infotrieve] |
3. | Clapham, D. E. (1995) Cell 80, 259-268[Medline] [Order article via Infotrieve] |
4. |
Parekh, A. B.,
and Penner, R.
(1997)
Physiol. Rev.
77,
901-930 |
5. | Barritt, G. J. (1999) Biochem. J. 337, 153-169[CrossRef][Medline] [Order article via Infotrieve] |
6. | Casteels, R., and Droogmans, G. (1981) J. Physiol. (Lond.) 317, 263-279[Abstract] |
7. | Gibson, A., McFadzean, I., Wallace, P., and Wayman, C. P. (1998) Trends Pharmacol. Sci. 19, 266-269[CrossRef][Medline] [Order article via Infotrieve] |
8. | Hoth, M., and Penner, R. (1992) Nature 355, 353-356[CrossRef][Medline] [Order article via Infotrieve] |
9. | Friel, D. D. (1996) Cell 85, 617-619[Medline] [Order article via Infotrieve] |
10. |
Montell, C.
(1997)
Mol. Pharmacol.
52,
755-763 |
11. | Arnon, A., Hamlyn, J. M., and Blaustein, M. P. (2000) Am. J. Physiol. 278, C163-C173 |
12. | Wayman, C. P., McFadzean, I., Gibson, A., and Tucker, J. F. (1996) Br. J. Pharmacol. 117, 566-572[Abstract] |
13. | Wayman, C. P., Wallace, H. M., Gibson, A., and McFadzean, I. (1999) Eur. J. Pharmacol. 376, 325-329[CrossRef][Medline] [Order article via Infotrieve] |
14. | Iwasawa, K., Nakajima, T., Hazama, H., Goto, A., Shin, W. S., Toyo-oka, T., and Omata, M. (1997) J. Physiol. (Lond.) 503, 237-251[Abstract] |
15. |
Iwamuro, Y.,
Miwa, S.,
Zhang, X. F.,
Minowa, T.,
Enoki, T.,
Okamoto, Y.,
Hasegawa, M.,
Furutani, H.,
Okazawa, M.,
Ishikawa, M.,
Hashimoto, N.,
and Masaki, T.
(1999)
Br. J. Pharmacol.
126,
1107-1114 |
16. |
Trepakova, E. S.,
Csutora, P.,
Marchase, R. B.,
Cohen, R. A.,
and Bolotina, V. M.
(2000)
J. Biol. Chem.
275,
26158-26163 |
17. | Bolotina, V. M., Weisbrod, R. M., Gericke, M., Taylor, P., and Cohen, R. A. (1997) Biophys. J. 72, 336 (abstr.) |
18. | Trepakova, E. S., Csutora, P., Gericke, M., Marchase, R. B., Cohen, R. A., and Bolotina, V. M. (2000) Biophys. J. 78, 193 (abstr.) |
19. |
Hirakawa, Y.,
Gericke, M.,
Cohen, R. A.,
and Bolotina, V. M.
(1999)
Am. J. Physiol.
277,
H1732-H1744 |
20. |
Cohen, R. A.,
Weisbrod, R. M.,
Gericke, M.,
Yaghoubi, M.,
Bierl, C.,
and Bolotina, V. M.
(1999)
Circ. Res.
84,
210-219 |
21. | Hoth, M., Fasolato, C., and Penner, R. (1993) Ann. N. Y. Acad. Sci. 707, 198-209[Medline] [Order article via Infotrieve] |
22. | Benham, C. D., Bolton, T. B., and Lang, R. J. (1985) Nature 316, 345-347[Medline] [Order article via Infotrieve] |
23. | Loirand, G., Pacaud, P., Mironneau, C., and Mironneau, J. (1986) Pflügers Arch. 407, 566-568[Medline] [Order article via Infotrieve] |
24. | Benham, C. D., and Tsien, R. Y. (1987) Nature 328, 275-278[CrossRef][Medline] [Order article via Infotrieve] |
25. | Benham, C. D., Hess, P., and Tsien, R. W. (1987) Circ. Res. 61, I-10-I-16 |
26. | Inoue, R., Kitamura, K., and Kuriyama, H. (1987) Pflügers Arch. 410, 69-74[Medline] [Order article via Infotrieve] |
27. |
Inoue, R.,
and Isenberg, G.
(1990)
Am. J. Physiol.
258,
C1173-C1178 |
28. | Chen, C., and Wagoner, P. K. (1991) Circ. Res. 69, 447-454[Abstract] |
29. | Loirand, G., Pacaud, P., Baron, A., Mironneau, C., and Mironneau, J. (1991) J. Physiol. (Lond.) 437, 461-475[Abstract] |
30. | Pacaud, P., and Bolton, T. B. (1991) J. Physiol. (Lond.) 441, 477-499[Abstract] |
31. | Komori, S., Kawai, M., Takewaki, T., and Ohashi, H. (1992) J. Physiol. (Lond.) 450, 105-126[Abstract] |
32. | Sims, S. M. (1992) J. Physiol. (Lond.) 449, 377-398[Abstract] |
33. | Wang, Q., Hogg, R. C., and Large, W. A. (1993) Pflügers Arch. 423, 28-33[Medline] [Order article via Infotrieve] |
34. | Guerrero, A., Fay, F. S., and Singer, J. J. (1994) J. Gen. Physiol. 104, 375-394[Abstract] |
35. | Hughes, A. D., and Bolton, T. B. (1995) Br. J. Pharmacol. 116, 2148-2154[Abstract] |
36. | Nakajima, T., Hazama, H., Hamada, E., Wu, S.-N., Igarashi, K., Yamashita, T., Seyama, Y., Omata, M., and Kurachi, Y. (1996) J. Mol. Cell. Cardiol. 28, 707-722[CrossRef][Medline] [Order article via Infotrieve] |
37. | Inoue, R., and Kuriyama, H. (1993) J. Physiol. (Lond.) 465, 427-448[Abstract] |
38. | Isenberg, G. (1993) EXS 66, 247-260[Medline] [Order article via Infotrieve] |
39. | Fasolato, C., Innocenti, B., and Pozzan, T. (1994) Trends Pharmacol. Sci. 15, 77-83[CrossRef][Medline] [Order article via Infotrieve] |
40. |
Trepakova, E. S.,
Cohen, R. A.,
and Bolotina, V. M.
(1999)
Circ. Res.
84,
201-209 |
41. | Yao, Y., Ferrer-Montiel, A. V., Montal, M., and Tsien, R. Y. (1999) Cell 98, 475-485[Medline] [Order article via Infotrieve] |
42. | Zweifach, A., and Lewis, R. S. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6295-6299[Abstract] |
43. | Petersen, C. C. H., Berridge, M. J., Borgese, M. F., and Bennett, D. L. (1995) Biochem. J. 311, 41-44[Medline] [Order article via Infotrieve] |
44. | Zhu, X., Jiang, M., Peyton, M., Boulay, G., Hurst, R., Stefani, E., and Birnbaumer, L. (1996) Cell 85, 661-671[Medline] [Order article via Infotrieve] |
45. | Zitt, C., Zobel, A., Obukhov, A. G., Harteneck, C., Kalkbrenner, F., Luckhoff, A., and Schultz, G. (1996) Neuron 16, 1189-1196[Medline] [Order article via Infotrieve] |
46. |
Xuan, Y.-T.,
Wang, O.-L.,
and Whorton, A. R.
(1992)
Am. J. Physiol.
262,
C1258-C1265 |
47. | Uyama, Y., Imaizumi, Y., and Watanabe, M. (1993) Br. J. Pharmacol. 110, 565-572[Abstract] |
48. | Kwan, C.-Y., Chaudhary, R., Zheng, X. F., Ni, J., and Lee, R. M. K. W. (1994) Hypertension 23, I-156-I-160 |
49. | Maggi, C. A., Giuliani, S., and Santicioli, P. (1995) Br. J. Pharmacol. 114, 127-137[Abstract] |
50. | Abe, F., Karaki, H., and Endoh, M. (1996) Br. J. Pharmacol. 118, 1711-1716[Abstract] |
51. | Sekiguchi, F., Shimamura, K., Akashi, M., and Sunano, S. (1996) Br. J. Pharmacol. 118, 857-864[Abstract] |
52. | Skutella, M., and Ruegg, U. T. (1996) Biochem. Biophys. Res. Commun. 218, 837-841[CrossRef][Medline] [Order article via Infotrieve] |
53. | Nomura, Y., Asano, M., Ito, K., Uyama, Y., Imaizumi, Y., and Watanabe, M. (1997) Br. J. Pharmacol. 120, 65-73[Abstract] |
54. | Smaili, S. S., Cavalcanti, P. M., Oshiro, M. E., Ferreira, A. T., and Jurkiewicz, A. (1998) Eur. J. Pharmacol. 342, 119-122[CrossRef][Medline] [Order article via Infotrieve] |
55. | Takemoto, M., Takagi, K., Ogino, K., and Tomita, T. (1998) Br. J. Pharmacol. 124, 1449-1454[Abstract] |
56. |
Tosun, M.,
Paul, R. J.,
and Rapoport, R. M.
(1998)
J. Pharmacol. Exp. Ther.
285,
759-766 |
57. | Guide for the Care and Use of Laboratory Animals (1996), National Academy of Sciences, Washington, D. C. |