Departments of Anesthesiology and Physiology and Biophysics, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905
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ABSTRACT |
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A -escin-permeabilized canine
tracheal smooth muscle preparation was used to test the hypothesis that
cGMP decreases Ca2+ sensitivity in
airway smooth muscle primarily by inhibiting the membrane
receptor-coupled mechanisms that regulate
Ca2+ sensitivity and not by
inhibiting Ca2+/calmodulin
activation of the contractile proteins. 8-Bromo-cGMP (100 µM) had no
effect on the free Ca2+
concentration-response curves generated in the absence of muscarinic receptor stimulation. In the presence of 100 µM ACh plus 10 µM GTP,
8-bromo-cGMP (100 µM) caused a rightward shift of the free Ca2+ concentration-response curve,
significantly increasing the EC50 for free Ca2+ from 0.35 ± 0.03 to 0.75 ± 0.06 µM; this effect of 8-bromo-cGMP was concentration
dependent from 1 to 100 µM. 8-Bromo-cGMP (100 µM) decreased the
level of regulatory myosin light chain (rMLC) phosphorylation for a
given cytosolic Ca2+ concentration
but had no effect on the amount of isometric force produced for a given
level of rMLC phosphorylation. These findings suggest that cGMP
decreases Ca2+ sensitivity in
canine tracheal smooth muscle primarily by inhibiting the membrane
receptor-coupled mechanisms that modulate the relationship between
cytosolic Ca2+ concentration and
rMLC phosphorylation.
guanosine 3',5'-cyclic monophosphate; trachea; calcium
sensitivity; myosin light chain phosphorylation; acetylcholine; guanosine
5'-O-(3-thiotriphosphate); fura
2; -escin; nitrovasodilators
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INTRODUCTION |
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NITRIC OXIDE (NO) has been implicated in a variety of important bioregulatory processes including normal physiological control of smooth muscle tone in blood vessels (16) and airways (11). Consequently, NO and compounds that generate NO (e.g., sodium nitroprusside) play an increasingly important therapeutic role in medicine by relaxing smooth muscle. However, despite the widespread use of nitrovasodilators and the recent introduction of inhaled NO therapy into clinical practice (32), the mechanism of action of NO on smooth muscle is not fully understood. It is known that NO relaxes airway smooth muscle in part by activating soluble guanylate cyclase and increasing cytosolic cGMP concentration ([cGMP]i) (13). It is thought that cGMP activates a cGMP-dependent protein kinase (6), which relaxes smooth muscle by phosphorylating specific substrates that not only reduce cytosolic Ca2+ concentration ([Ca2+]i), the focus of much previous work (13, 26), but also reduce the amount of force produced for a given [Ca2+]i (i.e., Ca2+ sensitivity) (23, 26, 31). Recent evidence indicates that cGMP-independent mechanisms also may be important (4).
In smooth muscle, agonist-induced contraction is mediated by an increase in [Ca2+]i, which binds calmodulin and subsequently increases myosin light chain kinase (MLCK) activity and phosphorylation of the 20-kDa regulatory myosin light chain (rMLC) (34), and by GTP-dependent, membrane receptor-coupled mechanisms that regulate Ca2+ sensitivity (7, 17-19, 21, 27). Although the mechanisms producing agonist-induced increases in Ca2+ sensitivity are not fully understood, studies of both permeabilized vascular (7, 16, 27) and nonvascular (19, 21) smooth muscles have demonstrated an increase in the levels of both rMLC phosphorylation and isometric force for a given [Ca2+]i. A GTP-binding regulatory protein (G protein)-mediated inhibition of the rMLC phosphatases may account for these observations (18, 35). Conversely, other studies (26, 28) suggested that Ca2+ sensitivity can be increased by receptor agonists independently of rMLC phosphorylation, such that the relationship between rMLC phosphorylation and force is regulated.
Possible mechanisms by which cGMP could decrease
Ca2+ sensitivity can be broadly
categorized into those that inhibit
Ca2+/calmodulin activation of MLCK
and the contractile proteins and those that inhibit the membrane
receptor-coupled mechanisms that regulate
Ca2+ sensitivity. These two
classes of mechanisms may have different sensitivities to cGMP. Prior
studies (15, 22, 29, 31, 37) of vascular and gut smooth muscles show
that cGMP and 8-bromo-cGMP, an analog of cGMP not susceptible to
metabolism by phosphodiesterases, reduce
Ca2+ sensitivity at nanomolar
concentrations during Ca2+
activation of permeabilized preparations. Because of this action, it is
difficult to examine possible additional effects of cGMP on
agonist-induced increases in Ca2+
sensitivity in these preparations. Although data are limited, studies
(3, 33) suggested that permeabilized airway smooth muscle activated by
Ca2+ alone may be relatively less
sensitive to the effects of cGMP. There are no data regarding the
effects of cGMP on agonist-induced Ca2+ sensitization in airway
smooth muscle. Accordingly, the aim of this study was to determine
whether 8-bromo-cGMP inhibits agonist-induced Ca2+ sensitization in canine
tracheal smooth muscle (CTSM). We used a -escin-permeabilized smooth
muscle preparation, seeking conditions in which 8-bromo-cGMP might
inhibit agonist-induced Ca2+
sensitization but not Ca2+
activation in the absence of receptor stimulation. To explore the
mechanisms responsible for these effects, we further examined the
action of 8-bromo-cGMP on the relationship between
Ca2+ concentration, rMLC
phosphorylation, and force during agonist stimulation.
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METHODS |
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Experimental Techniques
Tissue preparation. Mongrel dogs (15-23 kg) of either sex were anesthetized with an intravenous injection of pentobarbital sodium (30 mg/kg) and exsanguinated. A 10- to 15-cm portion of the extrathoracic trachea was excised and immersed in chilled physiological salt solution of the following composition (in mM): 110.5 NaCl, 25.7 NaHCO3, 5.6 dextrose, 3.4 KCl, 2.4 CaCl2, 1.2 KH2PO4, and 0.8 MgSO4. Fat, connective tissue, and the epithelium were removed with tissue forceps and scissors.Isometric force measurements. Muscle
strips (width 0.1-0.2 mm, length 3-5 mm, wet weight
50-100 µg) were mounted in a 0.1-ml quartz cuvette and
continuously superfused at 2 ml/min with the physiological salt
solution (37°C) aerated with 95%
O2-5%
CO2 (pH 7.4;
PO2
400 mmHg;
PCO2
39 mmHg). One end of the
muscle strip was anchored via stainless steel microforceps to a
stationary metal rod, and the other end was anchored via stainless
steel microforceps to a calibrated force transducer (model KG4,
Scientific Instruments, Heidelberg, Germany). During a 3-h
equilibration period, the length of the strip was increased after
repeated isometric contractions (of 2- to 3-min duration) induced by 1 µM acetylcholine (ACh) until the isometric force was maximal (optimal
length). Each strip was maintained at this optimal length for the
remainder of the experiment. These tissues produced maximal isometric
forces of 2.0-4.1 mN.
Permeabilization procedure. The
tissues were permeabilized with -escin as previously described by
our laboratory (1, 2). Muscle strips were superfused for 20 min with a
relaxing solution containing 100 µM
-escin (25°C). The
composition of the relaxing solution was 1 nM free
Ca2+, 7.5 mM MgATP, 4 mM EGTA, 20 mM imidazole, 1 mM dithiothreitol, 10 mM creatinine phosphate, and 0.1 mg/ml of creatinine phosphokinase. The pH was buffered to 7.1 with KOH;
ionic strength was kept constant at 0.20 M by adjusting the
concentration of potassium acetate. After exposure to
-escin, the
permeabilized muscle strips were washed with the relaxing solution
without
-escin for 10 min. The
Ca2+ ionophore A-23187 (10 µM)
was added to the relaxing solution and all subsequent experimental
solutions to deplete the intracellular Ca2+ stores. Solutions of varying
free Ca2+ concentrations were
prepared with the algorithm of Fabiato and Fabiato (5).
rMLC phosphorylation measurements.
rMLC phosphorylation was measured by two-dimensional gel
electrophoresis (12, 26). Another set of permeabilized muscle strips
from the same dogs was placed in 5-ml polyethylene vials and
flash-frozen by rapid immersion in a dry ice-acetone slurry containing
6% (wt /vol) trichloroacetic acid (80°C). These strips
were not maintained at optimal length (12); preliminary experiments
demonstrated that muscle length does not affect phosphorylation
measurements in permeabilized tissues (data not shown), a finding
consistent with a previous study (25) of permeabilized vascular smooth muscle. Then the frozen tissues were slowly thawed to room temperature, weighed, and transferred to glass-glass homogenizers containing a
homogenizing solution composed of 1% sodium dodecyl sulfate (SDS),
10% glycerol, and 20 mM dithiothreitol (1 mg dry weight of tissue/100
µl SDS homogenizing solution). Homogenates were centrifuged
(10°C) at 4,000 g (20 min) to
remove excess debris, and the supernatant was subjected to isoelectric
focusing (2% ampholytes, pH 4.5-5.4) followed by SDS-gel
electrophoresis (14% acrylamide). Quantitation of rMLC phosphorylation
was performed by scanning densitometry of the Coomassie blue-stained
gels. rMLC phosphorylation was calculated by integrating the
electrophoretic spot corresponding to the phosphorylated rMLC as a
fraction of the total integration of both the phosphorylated and
unphosphorylated rMLCs.
Experimental Protocols
Three experimental protocols were conducted, each on a separate group of permeabilized CTSM strips. For each protocol, each experiment was conducted on tissues obtained from the same animal. Isometric force was normalized to the force induced by 10 µM free Ca2+ as determined in each strip before each experiment, which was 81 ± 8% of the maximal isometric force induced by 10 µM ACh in the intact tissue (37°C) before permeabilization.Dependence on muscarinic-receptor stimulation. This protocol was conducted to determine whether the effect of 8-bromo-cGMP on Ca2+ sensitivity requires muscarinic-receptor stimulation. One pair of permeabilized muscle strips was superfused with relaxing solution containing 100 µM ACh plus 10 µM GTP to supramaximally activate the muscarinic receptor-coupled mechanisms that regulate Ca2+ sensitivity; the relaxing solution superfusing a second pair did not contain these compounds (free Ca2+ alone). The solutions superfusing one muscle strip of each pair also contained the cell-permeable, nonhydrolyzable form of cGMP, 8-bromo-cGMP (100 µM). Free Ca2+ concentration-response curves (1 nM to 10 µM) were then generated under these conditions for each permeabilized muscle strip.
Dependence on 8-bromo-cGMP concentration. This protocol was conducted to determine whether the effect of 8-bromo-cGMP on Ca2+ sensitivity depends on 8-bromo-cGMP concentration. Four permeabilized muscle strips were superfused with a solution containing 0.1 µM free Ca2+, which is the approximate [Ca2+]i in intact unstimulated CTSM cells (9). The solution superfusing three of the muscle strips also contained 1, 10, or 100 µM 8-bromo-cGMP; the solution superfusing the fourth strip did not contain 8-bromo-cGMP (control). ACh concentration-response curves (0.1-100 µM) were then generated under these conditions for each permeabilized muscle strip. All solutions contained 10 µM GTP.
Effects of 8-bromo-cGMP on rMLC phosphorylation and isometric force. This protocol was conducted to determine the effect of 8-bromo-cGMP on the relationships between the free Ca2+ concentration of the extracellular solution and rMLC phosphorylation and between rMLC phosphorylation and isometric force. For isometric force measurements, one set of four permeabilized muscle strips was mounted in the superfusion system. Three of the muscle strips were contracted with one of three concentrations of free Ca2+ (0.1, 0.6, or 1.0 µM) in the presence of 100 µM ACh plus 10 µM GTP for 20 min. The fourth strip was incubated with 100 µM 8-bromo-cGMP and then contracted with 0.6 µM free Ca2+ in the presence of 100 µM ACh plus 10 µM GTP.
For rMLC phosphorylation measurements, pilot studies were first conducted to determine the time course of the relationship between rMLC phosphorylation and isometric force during contractions induced by free Ca2+ alone or by free Ca2+ in the presence of ACh plus GTP. These studies demonstrated that during stimulation with 0.8 µM (approximately EC50) free Ca2+ alone, rMLC phosphorylation increased monotonically and was sustained throughout stimulation for 20 min. In contrast, during stimulation with 0.8 µM free Ca2+ in the presence of 100 µM ACh plus 10 µM GTP, rMLC phosphorylation initially increased to peak levels at 1-2 min that were significantly greater than those produced by 0.8 µM free Ca2+ alone, was sustained at these peak levels for 5 min, and then declined to sustained levels significantly above those induced by 0.8 µM free Ca2+ alone by 15 min. Contractions induced by free Ca2+ alone or by free Ca2+ in the presence of ACh plus GTP were sustained for the entire duration of stimulation; the contractions were significantly greater in the strips stimulated in the presence of ACh plus GTP (data not shown). On the basis of these pilot studies, the effect of 8-bromo-cGMP on the relationships between free Ca2+ and rMLC phosphorylation and between rMLC phosphorylation and isometric force were measured after stimulation for 2 (peak rMLC phosphorylation measurements) and 15 (steady-state rMLC phosphorylation measurements) min.
To conduct the studies, two sets of five permeabilized muscle strips were placed in 5-ml polyethylene vials. Four muscle strips from each set were stimulated with one of four concentrations of free Ca2+ [0.001 (baseline), 0.1, 0.6, or 1.0 µM] for 2 or 15 min in the presence of 100 µM ACh plus 10 µM GTP; the fifth muscle strip from each set was incubated with 100 µM 8-bromo-cGMP and then stimulated with 0.6 µM free Ca2+ in the presence of 100 µM ACh plus 10 µM GTP. The relationships between the free Ca2+ concentration of the extracellular bathing solution and rMLC phosphorylation and between rMLC phosphorylation and isometric force were constructed with values measured from the four permeabilized muscle strips not exposed to 8-bromo-cGMP (control). Then the values obtained from the permeabilized muscle strips exposed to 8-bromo-cGMP were plotted to determine whether either relationship was affected.
Statistical Analysis
Data are expressed as means ± SE; n is the number of dogs. Concentration-response curves were compared by nonlinear regression analysis as described by Meddings et al. (24). In this method, force (F) at any concentration (C) of drug was given by the equation F = FmC/(EC50 + C), where Fm represents the maximal (or minimal) isometric force and EC50 represents the concentration that produces half-maximal isometric force for that drug. Nonlinear regression analysis was used to fit the values of Fm and EC50 to the data for F and C for each condition studied. This method allows for comparisons of curves to determine whether they are significantly different and whether this overall difference can be attributed to differences in Fm, EC50, or both parameters. Differences in rMLC phosphorylation were assessed by analysis of variance, with post hoc analysis with the Student-Newman-Keuls procedure. The effect of 8-bromo-cGMP on rMLC phosphorylation induced by free Ca2+ during stimulation with ACh plus GTP was determined by unpaired Student's t-test. To determine whether 8-bromo-cGMP altered the amount of isometric force produced by a given level of rMLC phosphorylation, isometric force was calculated for the rMLC phosphorylation values measured during exposure to 8-bromo-cGMP by interpolation with a nonlinear polynomial regression of the measured control isometric force values. Then these isometric force values were compared by unpaired Student's t-test. A P value < 0.05 was considered significant. ![]() |
RESULTS |
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Dependence on Muscarinic-Receptor Stimulation
ACh plus GTP caused a leftward shift of the free Ca2+ concentration-response curves (Fig. 1, left), significantly decreasing the EC50 for free Ca2+ from 0.81 ± 0.05 to 0.35 ± 0.03 µM, which indicates an increase in Ca2+ sensitivity. ACh plus GTP also significantly increased maximal force produced by 10 µM free Ca2+ (Fig. 1, left). 8-Bromo-cGMP had no significant effect on the free Ca2+ concentration-response curves generated by free Ca2+ alone (EC50 values of 0.81 ± 0.05 µM for free Ca2+ alone vs. 0.86 ± 0.04 µM for free Ca2+ and 8-bromo-cGMP; Fig. 1, left). However, in the presence of ACh plus GTP, 8-bromo-cGMP caused a marked rightward shift of the free Ca2+ concentration-response curve (Fig. 1, left), significantly increasing the EC50 for free Ca2+ from 0.35 ± 0.03 to 0.75 ± 0.06 µM; this EC50 value was not significantly different from that produced by free Ca2+ alone (0.75 ± 0.06 vs. 0.81 ± 0.05 µM for free Ca2+ alone).
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Dependence on 8-Bromo-cGMP Concentration
Increasing the free Ca2+ concentration in the superfusate from 1 nM to 0.1 µM had no effect on baseline force. ACh caused a concentration-dependent increase in isometric force at constant [Ca2+]i, indicating a concentration-dependent increase in Ca2+ sensitivity (Fig. 1, right). 8-Bromo-cGMP (1, 10, and 100 µM) caused a concentration-dependent rightward shift of the ACh concentration-response curve (Fig. 1, right), increasing the EC50 for ACh from 2.2 ± 0.1 (control) to 2.9 ± 0.2, 3.7 ± 0.2, and 4.7 ± 0.2 µM, respectively. 8-Bromo-cGMP also caused a concentration-dependent decrease in maximal force induced by 100 µM ACh.Effects of 8-Bromo-cGMP on rMLC Phosphorylation and Isometric Force
Stimulation with 0.1, 0.6, or 1.0 µM free Ca2+ in the presence of ACh plus GTP for 2 or 15 min significantly increased rMLC phosphorylation levels (Fig. 2). This increase was concentration dependent when rMLC phosphorylation was measured at 2 min (Fig. 2, left) but not when measured at 15 min (Fig. 2, right). The level of rMLC phosphorylation was significantly greater when measured at 2 min than when measured at 15 min over the range of free Ca2+ concentrations studied. Therefore, the increase in rMLC phosphorylation produced by ACh was not sustained, even though [Ca2+]i was constant and contractions were stable. 8-Bromo-cGMP significantly inhibited the increase in rMLC phosphorylation induced by 0.6 µM free Ca2+ during stimulation with ACh plus GTP for 2 (Fig. 2, left) or 15 (Fig. 2, right) min. However, 8-bromo-cGMP did not change the amount of isometric force produced for a given level of rMLC phosphorylation regardless of the duration of stimulation (Fig. 3).
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DISCUSSION |
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The major findings of this study are that in -escin-permeabilized
CTSM 1) 8-bromo-cGMP inhibits the
increase in Ca2+ sensitivity
induced by muscarinic-receptor stimulation but has no effect on the
Ca2+-force relationship in the
absence of receptor stimulation and 2) during muscarinic-receptor
stimulation, 8-bromo-cGMP inhibits the increase in rMLC phosphorylation
induced by free Ca2+ but does not
affect the isometric force produced for a given level of rMLC phosphorylation.
Permeabilized preparations have been used extensively to investigate
mechanisms of agonist-induced increases in
Ca2+ sensitivity in both vascular
and nonvascular smooth muscles (7, 8, 17, 19, 20, 26-28). The most
commonly used compounds to create these preparations are
Staphylococcus -toxin and
-escin (26, 30). With these preparations,
[Ca2+]i
can be manipulated and held constant by controlling the
Ca2+ concentration of the
extracellular solution bathing the smooth muscle, the large
intracellular proteins necessary for contraction are preserved, and
coupling of membrane receptors to mechanisms that regulate
Ca2+ sensitivity remain intact and
can be activated. The ability to control
[Ca2+]i,
which is difficult to achieve in intact smooth muscle, is particularly
useful in the study of Ca2+
sensitivity. Akao et al. (1) and Bremerich et al. (2) have previously validated this technique in canine airway smooth muscle.
Ca2+ activation of smooth muscle increases MLCK activity and rMLC phosphorylation, thus increasing actomyosin ATPase activity and force. Although mechanisms mediating agonist-induced increases in Ca2+ sensitization are not fully understood, such sensitization is accompanied by further increases in rMLC phosphorylation (7, 17, 27). In all systems studied to date, this increase is produced by a GTP-dependent inhibition of rMLC phosphatase activity rather than by effects on MLCK (18). Recent evidence suggests that both heterotrimeric G proteins coupled to membrane receptors and monomeric G proteins in the cytoplasm may be intermediaries in this process. Protein kinase C has also been implicated in the pathway in some types of smooth muscle, although a recent study by our laboratory (2) does not support a role for protein kinase C in regulating Ca2+ sensitivity in airway smooth muscle. Sensitization by mechanisms independent of rMLC phosphorylation has also been proposed but remains to be proven (10, 36).
8-Bromo-cGMP did not affect Ca2+
sensitivity in the absence of muscarinic-receptor stimulation,
indicating no direct effect of 8-bromo-cGMP, even at high
concentrations, on the cellular events modulated by
Ca2+/calmodulin activation of MLCK
or on the contractile proteins in CTSM. In contrast, several previous
studies (15, 22, 29, 31, 37) have found that cGMP and its analogs
inhibit force produced by free
Ca2+ alone in vascular and gut
smooth muscles. This action has been attributed to a direct activation
of rMLC phosphatase activity by cGMP-dependent protein kinase (22, 37).
Although few studies in airway smooth muscle are available, Savineau
and Marthan (33) found that 50 µM cGMP inhibited
Ca2+-activated force in
-escin-permeabilized human bronchial smooth muscle. Lee et al. (22)
have shown that vigorous permeabilization can eliminate the effects of
cGMP on Ca2+-induced contractions
in vascular smooth muscle, suggesting loss of a soluble factor such as
a cGMP-dependent protein kinase during the permeabilization process.
However, in our permeabilized CTSM, receptor-coupled mechanisms
regulating Ca2+ sensitivity were
quite susceptible to cGMP-mediated inhibition, suggesting that
substrates for cGMP were still present. Thus there would appear to be
pronounced differences between species and type of smooth muscle in the
sensitivity of Ca2+-activated
force (in the absence of receptor stimulation) to cGMP. This factor may
explain, in part, why inhaled NO and other compounds that increase
[cGMP]i appear to be
more potent in relaxing pulmonary arterial smooth muscle compared with
airway smooth muscle (14).
Our data suggest that 8-bromo-cGMP decreases Ca2+ sensitivity in CTSM primarily by inhibiting the membrane receptor-coupled mechanisms that regulate Ca2+ sensitivity. Because cGMP has a significant effect on Ca2+-activated force in the absence of receptor stimulation in most other smooth muscle preparations, there has heretofore been little opportunity to study the effects of cGMP on agonist-induced Ca2+ sensitization. For example, Wu et al. (37) found that 8-bromo-cGMP inhibited increases in force and rMLC phosphorylation produced by guanosine 5'-O-(3-thiotriphosphate) in permeabilized rabbit ileum at constant Ca2+. This finding suggests that cGMP can interfere with G protein regulation of rMLC phosphatase. However, interpretation of their finding is limited by the fact that 8-bromo-cGMP also inhibited increases in force and rMLC phosphorylation produced by Ca2+ alone in their tissue. Thus it is difficult to partition its effects between direct actions on rMLC phosphatase (important during activation with Ca2+ alone) and effects on the G protein pathways inhibiting rMLC phosphatase during agonist stimulation. Because 8-bromo-cGMP did not affect force produced by activation with Ca2+ alone in our preparation, its action was solely on GTP-dependent pathways mediating ACh-induced Ca2+ sensitization. The finding that it inhibited the increase in rMLC phosphorylation produced by a given Ca2+ concentration is consistent with an action on the systems that inhibit rMLC phosphatase activity in response to receptor stimulation. cGMP could interfere with the coupling of the heterotrimeric G protein to the muscarinic receptor, inhibit the activation of heterotrimeric or monomeric G proteins, or block the action of activated G proteins to inhibit rMLC phosphatases.
We also examined the effect of 8-bromo-cGMP on the relationship between rMLC phosphorylation and force to determine whether it might uncouple force from rMLC phosphorylation. A previous study (23) of intact swine carotid artery maximally stimulated with histamine demonstrated that sodium nitroprusside and nitroglycerin, compounds that increase [cGMP]i, produced substantially more relaxation than was expected by the decrease in rMLC phosphorylation. The authors concluded that nitrovasodilators relax vascular smooth muscle in part by decreasing the amount of force produced for a given level of rMLC phosphorylation. We found that 8-bromo-cGMP decreased the level of rMLC phosphorylation for a given [Ca2+]i during ACh stimulation but had no effect on the amount of isometric force produced for a given level of rMLC phosphorylation. Thus we found no evidence that cGMP affects receptor-linked systems that might regulate force independently of rMLC phosphorylation. Lee et al. (22) also found that cGMP had no effect on this relationship during activation with Ca2+ alone.
In conclusion, 8-bromo-cGMP decreases
Ca2+ sensitivity in
-escin-permeabilized CTSM by inhibiting the membrane
receptor-coupled cellular mechanisms that regulate
Ca2+ sensitivity and not by
inhibiting Ca2+/calmodulin
activation of MLCK or the contractile proteins as reported in other
types of smooth muscles. This effect can be explained entirely by
changes in rMLC phosphorylation without invoking mechanisms that
uncouple force from rMLC phosphorylation. This action on
receptor-linked pathways that regulate
Ca2+ sensitivity is a potentially
important mechanism by which nitrovasodilators relax airway smooth muscle.
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ACKNOWLEDGEMENTS |
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We thank R. Lorenz and K. Street for expert technical assistance.
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FOOTNOTES |
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This study was supported in part by National Heart, Lung, and Blood Institute Grants HL-54757 and HL-45532.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: K. A. Jones, Mayo Clinic, 200 First St. SW, Rochester, MN 55905.
Received 17 June 1998; accepted in final form 16 September 1998.
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