Department of Pharmacology and Toxicology, University of
Mississippi Medical Center, Jackson, Mississippi
Cyclic GMP relaxes swine tracheal smooth muscle. Relaxation occurs
because of decreases in intracellular calcium concentration ([Ca++]i) that are thought to occur through
hyperpolarization which inhibits calcium influx. Activation of
K+ channels has been suggested as the underlying mechanism
for the hyperpolarization. In the present study, the effects of
8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP, a
membrane-permeable analog of cyclic GMP) on acetylcholine
(ACh)-induced increases in [Ca++]i were
examined by laser scanning confocal microscopy in fluo 3-loaded single
cells. Membrane potential and currents were measured by the
perforated-configuration of patch-clamp method. 8-Bromo-cGMP (1 µM-0.1 mM) inhibited 0.1 µM ACh-induced oscillations in
[Ca++]i in a concentration-dependent manner.
Spontaneous changes in membrane potential were observed by the
patch-clamp method. Acetylcholine (0.03 µM) did not affect the
time-averaged mean potential. The spontaneous changes in membrane
potential were reduced and the cells were depolarized by 0.1 µM ACh
and to a greater degree by 1 µM ACh. This result is consistent with
previous observations of ACh-induced depolarization in intact tissue.
The application of 0.1 mM 8-Br-cGMP had no significant effects on
spontaneous changes in membrane potential and did not induce changes in
membrane potential in cells treated with 0.1 µM ACh. In
voltage-clamped cells, ACh (0.1 µM) induced oscillations in
calcium-activated K+ currents. 8-Bromo-cGMP (0.1 mM)
inhibited these ACh-induced oscillations in currents, but had no
significant effects on spontaneous changes in membrane current in
unstimulated cells. These data indicate that 8-Br-cGMP inhibits
ACh-induced increases in [Ca++]i by
mechanisms other than regulation of membrane potential.
 |
Introduction |
An
increase in intracellular calcium concentration is necessary for
agonist-induced contractions of tracheal smooth muscle. Elevation in
[Ca++]i triggers myosin
ATPase activation and cross-bridge cycling that leads to smooth muscle
contraction (Miller-Hance et al., 1988
; Somlyo and Somlyo,
1994). In fact, the degree of ACh-induced tension in swine tracheal
smooth muscle is correlated with the magnitude of the initial rise in
[Ca++]i (Shieh et
al., 1991, 1995).
Acetylcholine-induced elevation in
[Ca++]i consists of two
components. Binding of ACh to its receptor activates G proteins that stimulate PLC and activated PLC degrades phosphatidylinositol 4,5-bisphosphate into IP3 and diacylglycerol
(Berridge and Irvine, 1984). Inositol 1,4,5-trisphosphate induces an
initial burst of calcium release from intracellular stores by binding
to the IP3 receptor/channels located in the
sarcoplasmic reticulum membrane. Then, a sustained elevation of
[Ca++]i is observed in
muscle strips during continuous application of agonist (Shieh et
al., 1991, 1995). Extracellular calcium influx is involved in this
sustained rise in [Ca++]i
(Bourreau et al., 1993; Liu and Farley, 1996a; Tomasic
et al., 1992), and in the case of ACh-induced increases in
[Ca++]i, calcium influx
is believed to occur mainly through VDCC at ACh concentrations less
than 1 µM (Liu and Farley, 1996a; Shieh et al., 1995). The
activity of VDCC depends on the depolarization of cells and thus
reducing cell depolarization may limit the activation of VDCC and the
increase in [Ca++]i.
Two cyclic nucleotide second messengers (cAMP and cGMP) decrease
[Ca++]i in airway smooth
muscle (Felbel et al., 1988; Nuttle and Farley, 1996).
Nitric oxide increases cGMP by stimulating guanylyl cyclase (Murad,
1994). Beta-adrenergic agonists, the primary therapeutic bronchodilators, increase cAMP by activating adenylyl cyclase (Torphy,
1994). Cyclic GMP activates cGMP-dependent protein kinase and cAMP
activates cAMP-dependent protein kinase (Francis and Corbin, 1994).
Several studies have shown the activation of BKCa by cyclic nucleotide-generating agonists (Bialecki and Stinson-Fisher, 1995; Kume et al., 1994; Yamakage et al., 1996).
These observations lead to the hypothesis that cyclic
nucleotide-generating agonists decrease
[Ca++]i by inhibiting
calcium influx through VDCC via hyperpolarization of the
smooth muscle cells (Kotlikoff, 1993; Miura et al., 1992). These studies, however, did not demonstrate that activation of BKCa actually results in hyperpolarization. In
addition, other studies have suggested a minor role for
hyperpolarization in beta-adrenergic agonist-induced smooth
muscle relaxation (Chiu et al., 1993; Cook et
al., 1993) and decreases in
[Ca++]i (Nuttle and
Farley, 1996).
In the present study, we examined the effects of ACh and 8-Br-cGMP on
membrane potential to investigate whether these agents regulate
[Ca++]i by modulating
membrane potential. Recordings of membrane potential in single cells
with the amphotericin perforated-patch whole-cell configuration of the
patch-clamp technique revealed that spontaneous changes in membrane
potential occur and ACh depolarizes cells in a concentration-dependent
manner. The application of 0.1 mM 8-Br-cGMP had no significant effect
on spontaneous changes in membrane potential and did not induce marked
changes in membrane potential in cells treated with 0.1 µM ACh.
Oscillatory increases in
[Ca++]i and outward
KCa currents were inhibited by 0.1 mM 8-Br-cGMP. Our data indicate that 8-Br-cGMP inhibits ACh-induced increases in
[Ca++]i by mechanisms
other than regulation of membrane potential.
| |
Methods |
Chemicals.
The fluorescent dye fluo-3 AM was purchased from
Molecular Probes Inc. (Eugene, OR). 8-Bromo-cGMP was purchased from
Research Biochemicals International (Natick, MA). All the enzymes for
cell dissociation, amphotericin and chemicals for the PSS and the
pipette solution were purchased from Sigma (St. Louis, MO). Fetal
bovine serum for the culture medium was purchased from HyClone
Laboratories Inc. (Logan, UT).
Solutions.
Physiological saline solution contained (in mM):
140, NaCl; 5, KCl; 1, CaCl2; 5.5, glucose; 10, HEPES. The pH was adjusted to 7.4 with NaOH. An amphotericin stock
solution of 60 mg/ml in DMSO was prepared daily and diluted to a final
concentration of 120 µg/ml in filtered pipette solution every 2 to 3 hr. The pipette solution contained (in mM): 45, K2SO4; 50, KCl; 10, NaCl;
10, HEPES; 50, mannitol (pH 7.2 with KOH). The culture medium consisted of Dulbecco's Modified Eagle's Medium/F12 (Sigma, D 8900)
supplemented with 18 mM sodium bicarbonate, 10% fetal bovine serum,
penicillin (100 units/ml) and streptomycin (100 µg/ml). Fluo-3 AM
stock solution (2 mM) was prepared in DMSO, kept at 
20°C and
diluted to 2 µM in PSS just before use.
Cell isolation.
Male pigs (Yorkshire white, 4-6 weeks old,
20-30 kg) were purchased from local suppliers and kept at the animal
facility for 3 to 5 days until use. They were anesthetized with 5%
isoflurane (Ohio Medical Products, Madison, WI) and sacrificed by
exsanguination. The trachea was removed and transported in PSS with
antibiotics. The smooth muscle was cleaned of epithelia, gland cells
and connective tissue at room temperature. The smooth muscle was then
minced and incubated with protease (0.5 mg/ml, type XIV, 6.6 units/mg solid) dissolved in 10 ml PSS for 1 hr at 37°C. The tissue was removed from the protease-containing solution by centrifugation at
100 × g for 10 min at room temperature and resuspended
in an enzyme solution consisting of collagenase (1 mg/ml, type I, 300 units/mg solid) and trypsin inhibitor (1 mg/ml, type II-S) dissolved in
10 ml PSS. The tissue was incubated at 37°C for 40 to 50 min in this
solution. The cells were pelleted twice by centrifugation for 10 min at
100 × g in PSS to wash the cells free of enzymes and
resuspended in PSS. Cells were then plated on glass coverslips for
patch-clamp recording and on glass-bottomed dishes (MatTek Corp.,
Ashland, MA) for confocal microscopy. After removing unattached cells
by gentle suction, cells were covered with culture medium. Cells were
placed in an incubator (37°C, 5% CO2) and used
within 2 days.
Confocal microscopy.
The culture medium was removed and the
cells were incubated with 2 µM fluo-3 AM dissolved in PSS for 30 min
at 37°C. Intracellular calcium concentration was measured with a
Noran Odyssey confocal microscope system (Middleton, WI) including a
Nikon Diaphot microscope (Garden City, NY) fitted with a 60× oil
immersion lens. Excitation and emission wavelengths used were 488 and
510 nm, respectively. The laser intensity was set to 8% of maximum,
and the photomultiplier amplification was set at 2,900 to 3,500 (4,096 maximum). A 100 µm confocal slit was used and 32 frame averaging with
a sampling rate of 1 per second was performed. The brightness over time
of selected cells was measured with MetaMorphTM
(Universal Imaging Corporation, West Chester, PA). Data were stored as
ASCII files and imported to Origin (Microcal Software Inc.,
Northampton, MA) for plotting and analysis. All experiments were
performed during continuous perfusion at room temperature.
Patch-clamp recordings.
Electrophysiological recordings of
membrane potential and currents were obtained with the amphotericin
perforated-patch configuration of the conventional patch-clamp
recording technique (Hamill et al., 1981). Cells attached to
glass coverslips were placed in the recording chamber that was perfused
continuously with PSS (flow rate approximately 2 ml/min) at room
temperature. Pipettes (3-5 megohms) were pulled from borosilicate
glass (Dagan Corp., FMG 15, Minneapolis, MN) and fire polished
immediately before filling with pipette solution. The tip of the
pipette was then dipped into amphotericin-free pipette solution for 10 sec to permit easier giga-seal formation. Recordings were obtained with
an Axopatch 200 B amplifier (Axon Instruments, Foster City, CA). Data
were digitized and sampled at 1 kHz with a DigiData 1200 interface (Axon Instruments) and stored by an IBM-compatible PC system with pCLAMP6 data acquisition software (Axon Instruments). Stored data were
analyzed with the pCLAMP module of Origin and Fetchan of pCLAMP6.
Data analysis.
Time-averaged mean potential was obtained by
integration of the membrane potential divided by the integration time,
with Fetchan. Mean potential was obtained during the last 30 sec of
each application period to avoid any delays in effect caused by the
solution change. The mean potentials from more than two groups were
compared by one-way repeated-measures ANOVA followed by Bonferroni's
or Students-Newman-Keuls method for multiple comparisons. The paired
t test was used to compare mean potential before and after
stimulation. Differences were considered to be statistically
significant at P < .05. Data are reported as the mean ± S.E., and n indicates number of cells tested. Data in table
1 were expressed as the mean ± S.D.
to show the variability of membrane potential in different cells.
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TABLE 1
Measurements of changes in membrane potential
Each value represents the mean ± S.D. from five different pigs
(n = 10 cells). The symbol * denotes a statistical
significance relative to the measurements before stimulation (* P < .05, one-way repeated-measures ANOVA followed by Bonferroni's
method).
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|
| |
Results |
Inhibition of ACh-induced increases in
[Ca++]i by
8-Br-cGMP.
Changes in
[Ca++]i caused by ACh
and/or 8-Br-cGMP were measured by laser scanning confocal microscopy.
Acetylcholine, at a concentration of 0.1 µM, increased
[Ca++]i as reported
previously (Liu and Farley, 1996a; Nuttle and Farley, 1996). As shown
in figure 1A, 0.1 µM ACh induced a
transient increase followed by sustained oscillations in
[Ca++]i in single
isolated tracheal smooth muscle cells. The effect of 8-Br-cGMP on the
ACh-induced increases in
[Ca++]i was examined in
cells treated with this submaximal concentration of ACh (0.1 µM) (Liu
and Farley, 1996a; Shieh et al., 1991). Increasing concentrations of 8-Br-cGMP (0.001-0.1 mM, fig. 1B) or 0.1 mM alone
(data not shown) was applied. As shown in figure 1B, 8-Br-cGMP inhibited ACh-induced increases in
[Ca++]i in a
concentration-dependent manner in a pattern similar to the inhibitory
effect of cAMP (Nuttle and Farley, 1996). The effect of 8-Br-cGMP was
observed within 1 min after its application. The response to ACh did
not recover quickly after removal of 8-Br-cGMP. In all the cells
exposed to 0.1 µM ACh (n = 11), 1 mM 8-Br-cGMP inhibited [Ca++]i
oscillations, whereas 1 µM 8-Br-cGMP reduced the frequency of
oscillations in 6 of 11 cells. In 22 of 24 cells, 0.1 mM 8-Br-cGMP inhibited oscillations, and thus this concentration was used in further
experiments to investigate the mechanism of
[Ca++]i inhibition.
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Fig. 1.
Effects of 8-Br-cGMP on ACh-induced increases in
[Ca++]i measured by laser scanning confocal
microscopy. (A) Acetylcholine (0.1 µM) induced oscillatory increases
in [Ca++]i. (B) Sequential application of
8-Br-cGMP (0.001-0.1 mM) inhibited 0.1 µM ACh-induced
[Ca++]i oscillations in a
concentration-dependent manner. Acetylcholine and 8-Br-cGMP were
applied at the times indicated by the arrows. Results shown are the
representative of data from three to five cells from three separate
cell isolations.
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Spontaneous changes in membrane potential and ACh-induced
depolarization.
The effects of ACh and 8-Br-cGMP on membrane
potential were investigated to test the hypothesis that these agents
regulate [Ca++]i by
modulating membrane potential. We used the perforated-patch method to
prevent the loss of cytosolic proteins and second messengers into the
pipette solution. Spontaneous changes in membrane potential as shown in
figure 2 were observed in all single cell
recordings. The time-averaged membrane potential was 25 ± 5 mV
(S.D., n = 10 cells from five different animals), with
a basal potential of 18 ± 4 mV and peak changes to 54 ± 8 mV. These spontaneous changes in membrane potential were inhibited by
ACh 
0.1 µM (fig. 2) and the cells were depolarized by 3 ± 2 mV (time-averaged mean membrane potential) with 0.1 µM ACh and
by 9 ± 4 mV with 1 µM ACh (table1).
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Fig. 2.
Spontaneous and ACh-induced changes in membrane
potential. Single tracheal smooth muscle cells show spontaneous changes
in membrane potential with a time-averaged mean of 25 ± 5 mV
(S.D., n = 10 cells from five isolations). No
significant changes were observed in 0.03 µM ACh-treated cells.
Acetylcholine inhibited spontaneous changes in membrane potential and
induced the cell to depolarize at concentrations of 0.1 and 1 µM.
Note that during 0.1 µM ACh, rhythmic changes in membrane potential
occurred. Acetylcholine solutions were applied at the times indicated
by the arrows.
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|
Effect of 8-Br-cGMP on the spontaneous and ACh-induced changes in
membrane potential.
The effect of 8-Br-cGMP on membrane potential
was examined in both ACh-treated and unstimulated cells. Acetylcholine
(0.1 µM) inhibited spontaneous changes in membrane potential and
induced depolarization (fig. 3A). As
shown in figure 3A, 0.1 µM ACh depolarized cells as long as ACh was
applied. The mean membrane potential after 4 min of ACh stimulation was
not significantly different from the mean potential after 1 min ACh
application (fig. 3B). Acetylcholine-induced changes in membrane
potential were not affected by 0.1 mM 8-Br-cGMP (fig.
4, A and B), a concentration that
inhibited ACh-induced increases in
[Ca++]i (fig. 1B).
8-Bromo-cGMP also had no significant effects on the spontaneous changes
in membrane potential (fig. 5, A and B).
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Fig. 3.
Changes in membrane potential by prolonged
application of 0.1 µM ACh. (A) Acetylcholine (0.1 µM) inhibited
spontaneous changes in membrane potential. The effect of 0.1 µM ACh
on membrane potential was sustained while ACh was applied. Note again
that oscillatory changes in membrane potential occurred during ACh
response. (B) The time-averaged mean membrane potential indicates
depolarization of cells by 0.1 µM ACh. Data were obtained for 11 cells from nine animals. Data were expressed as the mean ± S.E.
and compared by use of one-way repeated-measures ANOVA followed by
Bonferroni's method. *Statistical significance from basal mean
potential (*P < .05).
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Fig. 4.
8-Br-cGMP and membrane potential in ACh-treated
cells. (A) The application of 0.1 mM 8-Br-cGMP had no significant
effect on 0.1 µM ACh-induced changes in membrane potential. (B) The
mean potential was not changed significantly by 0.1 mM 8-Br-cGMP in 0.1 µM ACh-treated cells. Data were obtained for 10 cells from nine
animals. Statistical comparisons were performed using one-way
repeated-measures ANOVA followed by Student-Newman-Keuls method.
*Statistically significant difference from mean basal potential
(*P < .05). The arrows indicate periods when chemicals were
applied.
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Fig. 5.
8-Br-cGMP and membrane potential in unstimulated
cells. (A) No significant changes in spontaneous changes in membrane
potential were observed by 0.1 mM 8-Br-cGMP. (B) The mean potential was
compared by use of the paired t test. Data were not
significantly different after application of 8-Br-cGMP. Data were
obtained for seven cells from five animals. The arrows indicate periods
when chemicals were applied.
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Effect of 8-Br-cGMP on the outward currents.
Changes in
membrane currents were examined to further investigate other possible
mechanisms for the effects of 8-Br-cGMP on [Ca++]i. Cells were
voltage-clamped at the estimated equilibrium potential for
Cl
current, 23 mV, to measure changes in
outward current. Spontaneous transient outward currents (figs.
6A and 7)
were observed at a holding potential of 23 mV. The STOC are caused by
the activation of KCa (Saunders and Farley,
1991). The effects of 8-Br-cGMP on STOC and ACh-induced changes in
outward currents were examined. In amphotericin-perforated whole cells
where the cytosolic compartment was kept relatively intact, 0.1 µM
ACh inhibited STOC and induced large oscillations in outward current
(fig. 6A). Acetylcholine-induced oscillations were rhythmic compared
with the irregular burst pattern of STOC, and the amplitudes of the
ACh-induced oscillations were five to seven times larger than that of
STOC. As shown in figure 6C, 8-Br-cGMP (0.1 mM) inhibited ACh-induced
oscillations in outward current. 8-Bromo-cGMP (0.1 mM) did not induce
any apparent changes in STOCs in unstimulated cells (fig. 7). The
time-averaged mean currents before and after 0.1 mM 8-Br-cGMP
application were 13 ± 3 and 14 ± 5 pA (n = 5 from three animals), respectively, and not significantly different
from each other.
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Fig. 6.
Inhibition of ACh-induced oscillations in outward
current by 8-Br-cGMP. (A) Acetylcholine (0.1 µM) decreased the
frequency of STOC and induced larger oscillations in outward current.
(B) The mean frequency of ACh-induced oscillations in outward current
did not change significantly during 0.1 µM ACh application for 4 min.
Data were for six cells from three animals. Statistical comparisons
were performed using paired t test. (C) 8-Br-cGMP (0.1 mM) inhibited ACh-induced oscillations in outward current. (D) The mean
frequency in ACh-induced oscillations was decreased significantly by
0.1 mM 8-Br-cGMP. Data were obtained for 10 cells from six animals.
Comparisons were performed by the one-way repeated-measures ANOVA
followed by the Student-Newman-Keuls method. Cells were voltage-clamped
at 23 mV, the estimated chloride equilibrium potential. The arrows
indicate the periods while chemicals were applied.
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Fig. 7.
8-Br-cGMP and STOC. No significant effect on STOC
by 8-Br-cGMP was observed. The currents were not changed by 8-Br-cGMP
(see text). Similar data were obtained for five cells from three
animals. A paired t test was performed for statistical
comparisons.
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| |
Discussion |
Spontaneous changes in membrane potential.
Many types of
smooth muscle including swine tracheal smooth muscle cells have STOC
and spontaneous transient inward currents (Benham and Bolton, 1986;
Janssen and Sims, 1994; Saunders and Farley, 1991, 1992). These are
believed to occur because of spontaneous release of calcium from
intracellular stores and activation of BKCa and
ClCa, respectively (Bolton and Imaizumi, 1996;
Janssen and Sims, 1994; Nelson et al., 1995; Saunders and
Farley, 1991). In this study, spontaneous changes in membrane potential
were shown in single isolated tracheal smooth muscle cells. The
spontaneous changes in membrane potential appear to be correlated with
STOC and spontaneous transient inward currents. Activation of transient outward currents should cause transient hyperpolarizations to the
K+ equilibrium potential (84 mV in this study).
However, the transient hyperpolarizations do not reach
K+ equilibrium potential, probably because both
KCa and ClCa are activated
during the spontaneous release of calcium from the sarcoplasmic reticulum that drives this response. The Cl
equilibrium potential is about 23 mV, thus the hyperpolarization should reach a potential between 23 and 84 mV.
Time-averaged membrane potential in single tracheal smooth muscle cells
was 25 ± 5 mV with use of perforated patch recording. However,
the range in a single cell was 18 to 54 mV (table 1). In intact
muscle strips, membrane potential at 37°C is about 60 mV in canine
and swine trachea (Farley and Miles, 1977; Murali Mohan et
al., 1988; Shieh et al., 1992) and 50 mV in human
trachea (Honda and Tomita, 1987). The membrane potential recorded in
intact tissue is generally quite stable. However, this potential
represents the average membrane potential across many cells connected
electrically through gap junction channels. The length constant of the
muscle is 3 to 4 mm (H.-M. H. Saunders and J. M. Farley,
unpublished observations), but the lengths of smooth muscle cells are
only 75 to 100 µm. Thus the potentials of individual cells are
modulated by a large volume of tissue. This effect will moderate
changes in potential in any single cell. Because of the random nature of the spontaneous currents in each cell, the spatially averaged membrane potential of all cells is stable. Other studies with enzyme-dissociated smooth muscle cells have reported somewhat lower
membrane potentials of about 35 mV in human bronchi (Janssen, 1996)
and 40 mV in guinea pig trachea (Nakajima et al., 1995). Another possible explanation for the lack of transient spontaneous changes in membrane potential in intact tissue is the minor
contribution of the BKCa to the resting membrane
potential in intact tissue. Previous studies with charybdotoxin, an
inhibitor of BKCa, did not increase resting
[Ca++]i or tension in
tracheal smooth muscle (Shieh et al., 1996). Voltage-dependent delayed rectifier channels have been suggested to be
the main channel responsible for maintaining resting membrane potential
in canine airway smooth muscle cells (Kotlikoff, 1990). In addition,
most membrane potential measurements in intact smooth muscle were made
at 37°C (Farley and Miles, 1977; Murali Mohan et al.,
1988; Shieh et al., 1992).
Effects of 8-Br-cGMP on ACh-induced changes in membrane
potential.
It has been reported that calcium influx is required to
maintain resting [Ca++]i
or ACh-induced increases in
[Ca++]i in smooth muscle
cells (Liu and Farley, 1996b; Shieh et al., 1991, 1995). In
swine tracheal smooth muscle, calcium influx is mediated by VDCC (Liu
and Farley, 1996a; Shieh et al., 1995; Tomasic et
al., 1992) and other non-voltage-mediated calcium influx (Liu and
Farley, 1996a; Shieh et al., 1995). Calcium influx through VDCC depends on the depolarization of cells and thus several hypotheses propose that changes in
[Ca++]i might be
accomplished by modulation of membrane potential. Earlier studies have
shown that cAMP and cGMP decrease
[Ca++]i (Felbel et
al., 1988; Nuttle and Farley, 1996; Rashatwar et al.,
1987), and activate BKCa (Bialecki and
Stinson-Fisher, 1995; Kume et al., 1994; Yamakage et
al., 1996). The activation of BKCa should
hyperpolarize cells leading to decreased activation of VDCC and calcium
influx. This has been a primary mechanism suggested for the action of
cAMP and cGMP-induced decreases in
[Ca++]i. However, we
found that 8-Br-cGMP did not change membrane potential significantly at
a concentration that inhibited ACh-induced increases in
[Ca++]i. These data are
consistent with a previous report that cAMP inhibited ACh-induced
oscillations in ClCa currents in cells under voltage-clamp at 80 mV (Nuttle and Farley, 1996). The fact that ACh-induced changes in membrane potential were unaffected during exposure of the cells to 8-Br-cGMP could be caused by several factors.
First, the activity of BKCa is reduced by
muscarinic receptor activation (Kotlikoff, 1993; Kume and Kotlikoff,
1991). Thus, activation of KCa by 8-Br-cGMP may
not overcome the inhibition. Second, ClCa
activity is increased by muscarinic receptor activation (Janssen and
Sims, 1992; Nuttle and Farley, 1996). This would tend to drive the cell
potential to the Cl equilibrium potential and
the increased conductance would decrease the relative importance of
K+ channel opening in membrane potential control.
Thus, the overall result is cell depolarization that is less sensitive
to KCa channel opening (Robertson et
al., 1993).
Effects of 8-Br-cGMP on the spontaneous changes in membrane
potential.
Cyclic GMP has been demonstrated to increase the
single-channel activity of BKCa (Robertson
et al., 1993; Yamakage et al., 1996). Robertson
et al. (1993) demonstrated that cGMP caused a 40- to 50-fold
increase in the open probability of BKCa at 10 mV. In our study, the average current carried by STOC at 23 mV was
not altered by 8-Br-cGMP, that is STOC amplitude was not greatly increased as would be predicted by Robertson et al. (1993).
This discrepancy may be explained partly by the fact that Nelson
et al. (1995) demonstrated that the STOC in the vascular
smooth muscle are completely inhibited by iberiotoxin indicating they
are caused solely by BKCa. In airway, STOC are
not totally inhibited by either charybdotoxin or iberiotoxin (J. Choi and J. M. Farley, unpublished observation). Saunders and
Farley (1991) suggested that STOC are composed of multiple types of
K+ channels. Thus, changes in the activity of
BKCa may not be sufficient to alter the amplitude
of STOC. In addition, the measurements of Robertson et al
(1993) were made with
[Ca++]i buffered to 300 nM. This is approximately the concentration estimated by Nelson
et al. (1995) to occur during a calcium spark associated
with STOC. In our studies,
[Ca++]i varied with time
during the STOC (or hyperpolarization). How this difference might be
important is not known.
Other possibilities for cyclic nucleotide-induced inhibition of
[Ca++]i, such as direct
modulation of calcium channels (Méry et al., 1991),
accelerated reuptake of calcium into stores (McGrogan et al., 1995), or inhibition of calcium release from stores
(Komalavilas and Lincoln, 1994) have been suggested but have not been
investigated further in detail.
In conclusion, the present study shows that 0.1 mM 8-Br-cGMP did not
change membrane potential significantly in untreated and 0.1 µM
ACh-treated cells. These data suggest that ACh-induced increases in
[Ca++]i are inhibited by
8-Br-cGMP by mechanisms other than regulation of membrane potential.
Further investigations are needed to define mechanisms to understand
how [Ca++]i is regulated
by 8-Br-cGMP.
The authors thank Joe Ed Smith for building perfusion systems
for confocal microscopy and patch clamp. We also thank Dr. Louise Nuttle for her helpful comments.
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Abstract
Introduction
-Adrenoceptor subtypes and the opening of plasmalemmal K+-channels in bovine trachealis muscle: studies of mechanical activity and ion fluxes.
Br J Pharmacol
109:
1149-1156[Medline].
