Department of Internal Medicine II, Technical University of Munich, 81675 Munich, Germany
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ABSTRACT |
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We investigated
the role of K+ channels and
intracellular Ca2+ stores in the
relaxations induced by the NO donor 3-morpholinosydnonimine (SIN-1) and
8-bromo-cGMP (8-BrcGMP), 8-(4-chlorophenylthio)-cGMP (pCPT-cGMP), and
,
-methylene-ATP in isolated segments of rat ileum.
The inhibitory responses to SIN-1 and the cGMP analogs were not
influenced by the K+ blockers
apamin, charybdotoxin, iberiotoxin, or glibenclamide, whereas
relaxations induced by
,
-methylene-ATP were abolished by apamin
and tetraethylammonium. The NO-donor SIN-1 and the cGMP analogs were
able to inhibit contractions induced by activation of L-type
Ca2+ channels (BAY-K-8644), by
carbachol (CCh), and by cyclopiazonic acid (CPA), a blocker of
sarcoplasmic Ca2+-ATPase. However,
the inhibition of the combined CPA and CCh response was reduced and the
dose-response curve of SIN-1 shifted to the right. Intracellular
Ca2+ stores were emptied by
incubation in Ca2+-free buffer and
repetitive stimulation with CCh or BAY-K-8644. After restoration of
extracellular Ca2+, the inhibitory
effect of SIN-1 and pCPT-cGMP was only attenuated, whereas in the
additional presence of CPA, the inhibitory effect of SIN-1 was blocked
and the effect of 8-BrcGMP reduced. Thus depleting intracellular
Ca2+ stores attenuated the effect
of SIN-1 and 8-BrcGMP, suggesting an involvement of functional
Ca2+ stores.
apamin; charybdotoxin; iberiotoxin; cyclopiazonic acid
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INTRODUCTION |
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A SUBSTANTIAL PART of the neuromuscular regulation of the enteric nervous system is the nonadrenergic noncholinergic (NANC) inhibitory neurotransmission of gastrointestinal (GI) smooth muscle. There is strong evidence indicating that nitric oxide (NO) or a related NO-donating substance and ATP are major candidate transmitters of NANC innervation in the GI tract (18, 19, 30). Because NO is an unstable gaseous agent, NO donors such as glyceryl trinitrate, sodium nitroprusside, and 3-morpholinosydnonimine (SIN-1) (33) have been used to study the effects of NO. NO is known to activate soluble guanylate cyclase with a subsequent increase in cGMP levels (16), which in turn causes activation of G kinase (33). This can be achieved directly by the stable analogs of cGMP, 8-bromo-cGMP (8-BrcGMP) (4, 26) and the more membrane-permeable compound 8-(4-chlorophenylthio)-cGMP (pCPT-cGMP) (28). The mechanism of action of NO and cGMP to induce smooth muscle relaxation is not fully understood. Several possibilities are a matter of debate: 1) cell membrane hyperpolarization (31), 2) sequestration of Ca2+ with lowered cytosolic Ca2+ concentration ([Ca2+]i) (22), 3) reduced sensitivity of the contractile apparatus, i.e., of myosin light-chain kinase (23), and 4) reduced activation of second messengers involved in the excitatory pathway.
ATP is known to induce relaxation via hyperpolarization of the cell
membrane (19). ,
-Methylene-ATP has been shown to be a useful
stable ATP agonist (32).
Electrophysiological studies suggest that NO, similar to other inhibitory NANC mediators, also induces hyperpolarization of the cell membrane (10, 30). In the pulmonary artery, aorta, and tracheal muscle of the guinea pig, blockade of Ca2+-dependent K+ channels has been demonstrated to decrease responses to NO donors such as SIN-1 (5). In GH4C1 cells, activation of a cGMP-dependent protein kinase stimulated large- conductance Ca2+-dependent K+ channel activity (35). In 1994, Bolotina et al. (6) presented evidence that NO directly activated charybdotoxin-sensitive Ca2+ channels in cell-free membrane patches of rabbit aorta.
Hyperpolarization of the cell membrane and receptor-bound mechanisms are able to regulate [Ca2+]i, which is a principal regulator of contraction in smooth muscle (11). The [Ca2+]i concentration is regulated by the sarcoplasmic reticulum on the one hand and influx of Ca2+ via cell membrane-bound voltage-operated and receptor-operated Ca2+ channels on the other hand (11). Particularly via voltage-operated L-type channels will [Ca2+]i increase with depolarization and decrease with hyperpolarization (11). Cell membrane potential is known to be modulated to a great extent by the large family of K+ channels, some of them having particular dependence on [Ca2+]i in regulating intracellular Ca2+.
Thus the aims of the present study were to investigate 1) the role of K+ channels and 2) the role of sarcoplasmic Ca2+ pumps responsible for Ca2+ sequestration in intracellular Ca2+ stores in NO donor- and G kinase activator-induced responses.
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METHODS |
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The experimental model and protocol designed to investigate inhibitory responses were used as previously described (1). In brief, male Wistar rats (400-500 g) were killed by intraperitoneal injection of pentobarbital sodium (100 mg/kg). The terminal ileum was immediately removed, and six full-thickness gut segments were cut from it (length 1.5 cm). These were orientated longitudinally and attached to an isometric force transducer (Swegma force displacement transducer SG 4-500). The tissue was maintained in Krebs-Ringer-bicarbonate solution (KRS) (115.5 mM NaCl, 1.16 mM MgSO4, 1.16 mM NaH2PO4, 11.1 mM glucose, 21.9 mM NaHCO3, 2.5 mM CaCl2, and 4.16 mM KCl) gassed with 95% O2-5% CO2 at 37°C. A resting tension of 1 g was applied to the muscle before equilibrating for 30 min. Changes in tension were amplified by a Hellige coupler and recorded on a Rikadenki chart recorder.
At the beginning and the end of each experiment, the response to a
submaximal contracting dose of carbachol (CCh)
(106 M) [or the
activator of L-type Ca2+ channels
BAY-K-8644 (10
7 M)]
was tested as a control. CCh produced submaximal contractions; the
maximum response was obtained with
10
5 M CCh, but at a
concentration of 10
6 M the
plateau of the contractile response was more stable. BAY-K-8644 (10
7 M) produced maximal
contractions, whereas further increase of BAY-K-8644 concentration
produced no higher contractile response. CCh (or BAY-K-8644) was washed
out after 5 min by three consecutive washes at 5-min intervals. The
tissue was then incubated for 5 min in buffer before application of the
next stimulus.
SIN-1-, 8-Br-cGMP-, pCPT-cGMP-, and
,
-methylene-ATP-induced inhibition.
After washout and incubation, tissues were precontracted using CCh or
BAY-K-8644. When the CCh- or the BAY-K-8644-induced response had
reached a plateau, usually 30 s after addition of the stimulus, the
inhibitory mediator was added. Concentration-response curves were
constructed using separate applications of a single concentration of
the inhibitory mediator, with washouts in between. In separate
experiments, the inhibitory effect of a given concentration of the
inhibitor on CCh- or BAY-K-8644-induced contraction was tested for
stability by repeating this protocol two times. In these experiments,
SIN-1 was used at a concentration of 5 × 10
4 M, which was
approximately the EC50 of the dose
range tested. In time controls, the inhibitory effect of SIN-1,
pCPT-cGMP, and
,
-methylene-ATP remained stable for >3 h with
this protocol. As far as 8-BrcGMP is concerned, the response to a
second application of the inhibitor was significantly decreased up to 1 h after the first application. For this reason, 8-BrcGMP was applied
only once per single preparation in the following experiments. Each segment of rat ileum was used only for a single concentration-response curve for agonist or blocker, respectively.
Effect of K+
channel blockers on inhibitor-induced response.
After the initial stimulation with CCh, the inhibitory mediator was
added as described above, with the inhibitory response serving as a
control. After the respective washes, the blocker was added to the bath
5 min before stimulation with CCh. In the presence of the respective
blocker, the effect on the inhibitor-induced response was tested. SIN-1
was used at the concentration of 5 × 104 M, which was
approximately the EC50 of the dose
range tested. 8-BrcGMP (10
3
M) and
,
-methylene-ATP
(10
4 M) were used to
compare a similar degree of relaxation. After repeated washes, the next
concentration of the blocker was applied. Each segment was used only
for a single concentration-response curve of a single blocker.
Experiments using cyclopiazonic acid.
Application of cyclopiazonic acid (CPA), a blocker of the sarcoplasmic
Ca2+-ATPase, was used to test
participation of intracellular
Ca2+ stores. CPA
(105 M) induced a slow
contractile response in the unstimulated muscle and was used as
prestimulation similar to CCh or BAY-K-8644 to assess the inhibitory
response of SIN-1, the cGMP analogs, and
,
-methylene-ATP. Second
applications of CPA elicited only ~60% of the first contractile
response in 12 of 20 preparations. Therefore CPA was applied only once
per preparation, when CPA was used as the only contractile stimulus. In
contrast, when the tissue was stimulated with CPA and CCh, the
resulting contractile response remained stable in repeated time
controls for at least 2 h. In this set of experiments, the second
contractile stimulus (CCh) was applied 4 min after CPA. The inhibitory
mediator was added as soon as the CCh-induced response had reached a
plateau, usually 30 s after addition of this stimulus. A
concentration-response curve was constructed using separate
applications of a single concentration of the NO donor SIN-1 on
combined prestimulation with CPA
(10
5 M) and CCh
(10
6 M), which were added
as described above.
Ca2+-depleting
protocol.
In a different series of experiments, the tissue was incubated in a
Ca2+-free Krebs-Ringer buffer
containing 0.25 mM EGTA and CPA
(105 M) to empty
intracellular Ca2+ stores and
stimulated with CCh (10
6 M)
or BAY-K-8644 (10
7 M).
After washout with Ca2+-free
buffer containing CPA, this procedure was repeated twice until
application of CCh (or BAY-K-8644) had virtually no contractile effect.
After washout the tissue was incubated in
Ca2+-free Krebs-Ringer buffer
without EGTA. This buffer was prepared omitting
CaCl2. After application of CPA
and CCh (or BAY-K-8644), restoration of extracellular
Ca2+ caused a contraction, which
reached an instant plateau. The respective inhibitory agent was added
within <10 s after restoration of extracellular Ca2+. Two sets of controls were
performed. In the first control, an identical protocol was used, but
CPA was omitted (see Table 3). In the second, the contractile response
was tested, applying KRS instead of the inhibitory agent (see Fig. 4).
Data analysis and statistics.
For data analysis the contraction level induced by the stimulus before
the addition of the inhibitory mediator was determined as control. To
study the fast component of relaxation, we defined the inhibitory
response that reached a maximum within 20 s after application of SIN-1
and ,
-methylene-ATP analogs and within 40 s after application of
the cGMP analogs as the remaining contraction after application of the
inhibitory mediator. Contractile responses were given as absolute
values, and the inhibition was expressed as a percentage of the control
contraction. Data are given as means ± SE, and
n indicates the number of independent
observations in different muscle strips. Each protocol was repeated in
ileal segments of at least two different animal preparations. When a statistical difference of two means was determined, we performed a
paired two-tailed Student's t-test.
For comparisons of more than two means one-way ANOVA, followed by post
hoc test with Bonferroni correction for multiple comparisons, was
carried out to determine statistical difference. Values of
P < 0.05 were considered
significant.
Drugs.
The following drugs were used in this study. SIN-1 was obtained from
Cassella-Riedel (Frankfurt, Germany), and pCPT-cGMP was from Biolog
(Bremen, Germany). CCh and BAY-K-8644 were procured from Bayer
(Heidelberg, Germany), and iberiotoxin was from Research Biochemicals
(Natick, MA). Tetraethylammonium (TEA) was from Aldrich (Milwaukee,
MN), and glibenclamide was from Boehringer Mannheim. 8-BrcGMP,
,
-methylene-ATP, TTX, apamin, charybdotoxin, CPA, and EGTA were
all obtained from Sigma (Munich, Germany). The drugs were freshly
dissolved in saline and further diluted with KRS. Glibenclamide was
dissolved in N-methylformamide. The
drugs were added to the bath in microliter volumes, and experiments
were controlled for the effects of the drug solvents.
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RESULTS |
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Effect of SIN-1 on CCh- and BAY-K-8644-induced contraction.
The muscarinic agonist CCh
(106 M) caused a tonic
contractile response of the longitudinal smooth muscle. As soon as the
contractile response of CCh reached a plateau, application of SIN-1
(10
6 to 3 × 10
3 M) caused an
instantaneous and concentration-dependent relaxation. The maximal
inhibitory effect obtained at the highest concentration of SIN-1 tested
(3 × 10
3 M)
was 93.3 ± 3.8% of the contraction induced by CCh
(10
6 M) (Fig.
1A).
Repetitive application of SIN-1 in a single preparation revealed no
change in relaxation when the preparation was washed with KRS
periodically [n = 8;
not significant (NS)]. The approximate EC50 of 5 × 10
4 M was used to compare
the response of SIN-1 with the other inhibitors (Tables
1 and 2). The
Ca2+ channel activator BAY-K-8644
was used to stimulate smooth muscle cells directly by inward
Ca2+ current, which is independent
from muscarinic receptor activation. BAY-K-8644 caused a tonic
contraction of the longitudinal smooth muscle segments, which reached
25% of the contractile CCh
(10
6 M) response at an
optimum concentration of
10
7 M. Higher
concentrations of BAY-K-8644 caused a decrease in contractile response.
Application of SIN-1 (5 × 10
4 M) on the plateau of
the BAY-K-8644-induced response caused an instantaneous relaxation
reaching 100.2 ± 4.6% of the BAY-K-8644 response
(n = 8) (Table 1).
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Effect of 8-BrcGMP and pCPT-cGMP on CCh- and BAY-K-8644-induced
contraction.
Application of 8-BrcGMP
(105 to
10
3 M) and pCPT-cGMP
(10
6 to 3 × 10
4 M) on the plateau of
the CCh-induced response caused a sustained concentration-dependent
relaxation (Fig. 1, B and
C). The onset of action of pCPT-cGMP
appeared to be faster than that of 8-BrcGMP, which in some tissues took
>1 min to reach its maximal effect. The inhibitory effect obtained at
the highest concentration of 8-BrcGMP tested
(10
3 M) was 49.4 ± 6.6% of the contraction induced by CCh
(10
6 M)
(n = 12). Repetitive application of
8-BrcGMP (10
3 M) on a
single preparation revealed a marked tachyphylaxis (1st application:
relaxation 49.4%; 2nd application: 17% of the CCh-induced response)
(n = 9). For this reason 8-BrcGMP was
applied only once per preparation in the following experiments.
Application of 8-BrcGMP (10
3 M) on the plateau of
the BAY-K-8644-induced response caused a sustained relaxation reaching
100.0 ± 7.8% of the BAY-K-8644 response (n = 9; Table 1).
Effect of ,
-methylene-ATP on CCh-
and BAY-K-8644-induced contraction.
Application of
,
-methylene-ATP
(10
8 to
10
4 M) on the plateau of
the CCh-response caused an instantaneous concentration-dependent relaxation (Fig. 1D). The inhibitory
effect of
,
-methylene-ATP (10
4 M) was 53.1 ± 5.7% (n = 9) (Table 1). Repetitive
application of
,
-methylene-ATP
(10
4 M) on a single
preparation revealed no desensitization (1st application: relaxation
49.8%; 2nd application: 40.5% of the CCh-induced response; n = 9).
Effect of K+
channel blockers on SIN-1-, 8-BrcGMP-, and
,
-methylene-ATP-induced inhibition.
A variety of K+ channel blockers
were tested against SIN-1 and 8-BrcGMP to gain insight into a possible
K+ channel type being functional
during muscle relaxation. Apamin (10
7 M;
n = 9; NS), charybdotoxin
(10
7 M;
n = 9; NS), and iberiotoxin
(10
8 M;
n = 6; NS), specific blockers of
Ca2+-dependent
K+ channels, had no influence on
the effect of SIN-1 (5 × 10
4 M) and 8-BrcGMP
(10
3 M) (Table 2), as
reported previously for NO-dependent relaxations in the canine
ileocolonic junction (9). Iberiotoxin applied at higher concentrations
of up to 10
6 M failed to
exert any influence on the SIN-1-induced relaxation (Table 2).
Furthermore, neither the nonspecific
K+ channel blocker TEA
(10
4 to
10
1 M) nor glibenclamide
(10
5 M;
n = 8) (Table 2), a specific blocker
of ATP-dependent K+ channels,
modified the inhibitory action of SIN-1 and 8-BrcGMP. The inhibitory
response of
,
-methylene-ATP
(10
4 M) was unaffected by
charybdotoxin (10
7 M) and
glibenclamide (10
5)
(Table 2), whereas the
Ca2+-activated
K+ blocker apamin
(10
7 M) significantly
reduced the
,
-methylene-ATP-induced inhibition (Table 2).
Application of TEA (10
1 M)
induced a contractile response and reduced the subsequent CCh
contraction. Thus, in comparison with controls, a significant baseline
shift of contractile response occurred. Application of
,
-methylene-ATP
(10
4 M) on this contractile
response failed to exert any relaxation. Hence TEA
(10
1 M) abolished
the
,
-methylene-ATP
(10
4 M)-induced inhibition
(0.1 ± 1.4% of the contractile response; n = 8).
Effect of CPA on SIN-1-, pCPT-cGMP-, and
,
-methylene-ATP-induced responses.
CPA (10
5 M), which is known
to block the sarcoplasmic
Ca2+-ATPase (34) and thereby to
increase
[Ca2+]i,
caused a slowly developing tonic contraction that reached a plateau and
increased to 15.1 ± 2.2 mN (n = 20). A second administration of CPA revealed a decrease to 60% of the
first contractile response in 12 of 20 preparations. Therefore CPA was
applied only once as a single contractile stimulus. Application of
SIN-1 (10
3 M) or pCPT-cGMP
(10
4 M) on this plateau
caused a prompt relaxation, reaching 41.2 ± 4.4% (SIN-1;
n = 15) or 88.2 ± 5.7%
(pCPT-cGMP; n = 11) of the CPA-induced
response (Fig. 2). Because of the
attenuation of the SIN-1-induced inhibition in the presence of CPA, for
the following experiments
10
3 M SIN-1 was used.
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Effect of CPA on SIN-1- and pCPT-cGMP-induced responses after
prestimulation with CCh.
Preincubation with CPA (105
M) and subsequent application of CCh
(10
6 M) induced a combined
contractile response that did not exceed the contractile response of
CCh (10
6 M) alone (35.1 ± 5.3 vs. 36.3 ± 4.6 mN, n = 8) (Fig. 2). When a concentration-response curve of SIN-1 was carried
out on this combined contractile response, the curve was shifted to the
right compared with control stimulation with CCh
(10
6 M) alone (Fig.
3). The inhibitory effect of SIN-1, applied
in a concentration of 10
3
M, was significantly reduced after combined stimulation with CPA and
CCh (39.5 ± 7.4% of combined contraction vs. 82.9 ± 5.8% of
CCh-induced contraction alone; P < 0.05; n = 8; Fig.
2A).
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Effect of depleting intracellular
Ca2+ stores on
SIN-1-, 8-BrcGMP-, and
,
-methylene-ATP-induced responses.
Intracellular Ca2+ stores were
depleted by repetitive stimulation of the smooth muscle with CCh
(10
6 M) in
Ca2+-free buffer prepared with
EGTA either in the presence or absence of CPA. After several cycles,
the medium was changed to
Ca2+-free buffer without EGTA in
the presence or absence of CPA
(10
5 M). Addition of CCh
(10
6 M) or BAY-K-8644
(10
7 M) under these
conditions elicited no response but induced an immediate contraction
after subsequent restoration of external Ca2+ (Fig.
4). The inhibitory effects of SIN-1,
8-BrcGMP, or
,
-methylene-ATP were tested immediately after the
contraction had reached a plateau level.
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DISCUSSION |
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Application of SIN-1 on longitudinal smooth muscle of rat ileum
precontracted with CCh caused a concentration-dependent relaxation. Contractions that involve protein kinase C (29) and intracellular Ca2+ stores have previously been
shown to be inhibited by NO donors and cGMP analogs (34). The
concentration range of the inhibitory action of SIN-1 is in good
agreement with other reports on the action of SIN-1 in ventricular
myocytes of the frog (21) and in isolated leukocytes (24). In the
above-mentioned studies (21, 24), there was a biphasic effect with an
excitatory portion at concentrations of SIN-1 in the nanomolar range. A
similar response to NO donors was reported in the rat small intestine
(3). The fact that we did not find any excitatory effect seems most
likely to be due to the submaximal prestimulation before SIN-1
application. The cell membrane-permeable and stable cGMP analogs
8-BrcGMP and pCPT-cGMP showed a concentration-dependent inhibitory
effect. The concentration range of the inhibitory action is in line
with other reports on the action of 8-BrcGMP in opossum esophageal longitudinal muscle (26) and in the canine pyloric sphincter (4).
Neural blockade by TTX (106
M) did not modify the SIN-1- and cGMP-induced relaxation, demonstrating an inhibitory action independent from nerves.
In GI smooth muscle there is ample evidence that NO-mediated
relaxations involve hyperpolarization (22, 31), and it is most likely
that this change in membrane potential is induced by
K+ channels, which are activated
via cGMP (17). Opening of these channels would hyperpolarize the
membrane and relax smooth muscle. However, our results show that apamin
(107 M), charybdotoxin
(10
7 M), iberiotoxin
(10
6 M), and glibenclamide
(10
5 M), known blockers of
Ca2+- and ATP-dependent
K+ channels, and TEA
(10
1 M) were without effect
on the SIN-1- or the 8-BrcGMP-induced relaxation of rat ileum
longitudinal muscle. These results are in clear contrast
to several reports (5, 25) on vascular and tracheal smooth muscle that
cGMP-dependent protein kinases activate
Ca2+-dependent
K+ channels.
Thus the inhibitory response induced by NO donors and cGMP-dependent mechanisms does not depend on K+ channels sensitive to the blockers tested. However, the ineffectiveness of all the K+ channel blockers used in our experiments does not exclude a role of other K+ channels for the effect of SIN-1 and cGMP analogs in this tissue, since it has been reported that NO-induced K+ channel activation in GI smooth muscle is not affected by any of the specific blockers used in this study (8, 14, 17).
The inhibitory effect of ,
-methylene-ATP, a stable analog of ATP,
has been described previously (32). In contrast to SIN-1 and 8-BrcGMP,
the inhibitory response of
,
-methylene-ATP was blocked by apamin
and TEA, indicating that different cellular mechanisms were involved. A
similar influence of apamin on ATP-induced inhibition has been reported
in previous studies (2) and is thought to support a participation of
Ca2+-dependent
K+ channels in mediating
ATP-induced relaxation. In patch-clamp experiments on smooth muscle of
chicken rectum,
,
-methylene-ATP induced an inward current
followed by a K+ outward current
(20). Both currents were unchanged after depletion of intracellular
stores with caffeine and blockade of L-type
Ca2+ channels with nifedipine.
Finally, removal of extracellular
Ca2+ abolished the outward current
(20).
To test the involvement of functional intracellular
Ca2+ stores, we used CPA, a
blocker of the Ca2+-ATPase of the
sarcoplasmic reticulum. Depleting intracellular Ca2+ stores with CPA and
Ca2+-free extracellular medium
attenuated the ,
-methylene-ATP-induced inhibition in the presence
of CCh. Consequently, CPA-dependent intracellular
Ca2+ stores participate in the
,
-methylene-ATP-induced relaxation.
CPA blocks the sarcoplasmic
Ca2+-ATPase and thereby is thought
to increase
[Ca2+]i
and induce contraction (34). Thapsigargin, another blocker of
sarcoplasmic Ca2+-ATPase, was not
used because of a postulated direct interaction with voltage-dependent
Ca2+ channels (7). The attenuation
and rightward shift of the SIN-1-induced effect on the combined
contractile response of CPA and CCh cannot be explained by a baseline
shift of precontraction, because CCh (106 M) caused a submaximal
response and the combined response of CPA plus CCh did not exceed the
contractile response of CCh alone. However, we cannot exclude a shift
of
[Ca2+]i
and/or intracellular protein kinases at this identical level of
contraction. As the combination of CCh prestimulation and presence of
CPA also reduced the inhibitory effect of the selective cGMP analog
pCPT-cGMP, it can be speculated that the cGMP-dependent mechanisms
inhibiting smooth muscle contraction are also dependent on functional
intracellular Ca2+ stores.
To investigate a possible involvement of intracellular Ca2+ changes, we used repetitive stimulation with CCh in Ca2+-free buffer in the presence and absence of CPA to deplete the intracellular Ca2+ stores.
After emptying the intracellular Ca2+ stores with CPA, which was present to block the sarcoplasmic Ca2+-ATPase, we found that the inhibitory effects of all three inhibitors were either significantly reduced or abolished compared with control conditions without depleting the Ca2+ stores. The fact that this difference in the inhibitory effect occurred in CCh- as well as in BAY-K-8644-prestimulated tissue suggests that intracellular Ca2+ stores seem to be of importance in the inhibitory responses elicited on BAY-K-8644-induced contraction. This finding supports the idea that even with pure stimulation of Ca2+ influx through L-type Ca2+ channels, the inhibitory effect of all three inhibitors involves functional internal Ca2+ stores, regardless of the Ca2+ source for the contraction. It is possible that CPA treatment depletes intracellular Ca2+ stores involved in both contractile and inhibitory responses. In contrast, other strategies used to deplete Ca2+ (e.g., use of Ca2+-free medium and repetitive stimulation with CCh or BAY-K-8644 in the absence of CPA) may selectively remove stores responsible for the contractile responses.
The inhibitory effect of 8-BrcGMP showed less sensitivity to depletion of intracellular Ca2+ stores than the inhibitory responses to SIN-1. This effect was more pronounced after prestimulation with CCh than with BAY-K-8644. In general, 8-BrcGMP showed a smaller inhibitory response compared with SIN-1, and the onset and time course of this inhibition was slower than the immediate inhibitory effect of SIN-1. This prolonged time course of the cGMP-induced inhibition may have led to partial refilling of intracellular Ca2+ stores, especially when the Ca2+-ATPase was not blocked by CPA. Additionally, it may be possible that the high concentration of 8-BrcGMP causes a small degree of cross-activation of other second messenger mechanisms, such as protein kinase A (15). This could explain why the inhibitory effect of 8-BrcGMP was not completely abolished after Ca2+ depletion in the presence of CPA.
Altogether, contractions induced by CPA alone were inhibited ~50% by SIN-1. This inhibition is significant and similar to the SIN-1-induced inhibition of CCh responses. The combined presence of CPA and CCh reduced the SIN-1-induced effect substantially, and SIN-1-responses were abolished after depleting the stores in Ca2+-free medium. Thus strategies such as the presence of CCh and exposure to Ca2+-free medium appear to be necessary in addition to CPA to reveal an attenuation or block of the SIN-1-induced inhibition.
Possible targets of SIN-1 and cGMP analogs in smooth muscle cells of rat ileum include the following: 1) K+ channels or other ion channels not sensitive to the blockers tested in this study; 2) the sarcoplasmic reticulum, in which sequestration of Ca2+ may be activated, thereby decreasing [Ca2+]i and causing relaxation (under this scheme, sequestration mechanisms would have to be at least partially CPA insensitive, because SIN-1 and pCPT-cGMP caused significant inhibition of CPA-induced contractions); 3) Ca2+ channels, in which influx of extracellular Ca2+ may be reduced, which has been reported for SIN-1 in cardiac muscle (21); 4) pathways independent of cytosolic Ca2+ as reported for sodium nitroprusside in vascular muscle by Sato et al. (27), when the tissue is relaxed by SIN-1 or the cGMP analogs. The relative role of these different mechanisms in GI muscle is not yet known.
We conclude that SIN-1 and cGMP analogs inhibit contractile responses to isolated muscarinic stimulation, isolated activation of Ca2+ influx via L-type channels (BAY-K-8644), and isolated blockade of sarcoplasmic Ca2+-ATPase (CPA). K+ channels sensitive to apamin, charybdotoxin, iberiotoxin, and glibenclamide are not involved in mediation of the relaxation. Depletion of intracellular Ca2+ stores in the presence of CPA attenuated the effect of the inhibitors substantially, suggesting an involvement of functional sarcoplasmic reticulum Ca2+ stores.
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ACKNOWLEDGEMENTS |
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We acknowledge the courtesy of Cassella-Riedel GMBH (Frankfurt, Germany). We thank C. Fleischer for laboratory work, L. Kots for proofreading the manuscript, and Dr. C. W. R. Shuttleworth and Dr. S. D. Koh for numerous discussions.
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FOOTNOTES |
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A portion of this study was presented at the annual meeting of the American Gastroenterological Association in San Diego, CA, on May 14-17, 1995, and has been published previously in abstract form (see Ref. 12).
Address for reprint requests: H.-D. Allescher, II. Medizinische Klinik und Poliklinik der TU München, Ismaningerstr. 22, 81675 München, Germany.
Received 26 February 1997; accepted in final form 25 March 1998.
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