Departments of Anesthesiology and Physiology and Biophysics, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905
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ABSTRACT |
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Muscarinic receptor stimulation increases
Ca2+ sensitivity, i.e., the amount
of force produced at a constant submaximal cytosolic Ca2+ concentration
([Ca2+]i),
in permeabilized smooth muscle preparations. It is controversial whether this increase in Ca2+
sensitivity is in part mediated by protein kinase C (PKC). With the use
of a -escin permeabilized canine tracheal smooth muscle (CTSM)
preparation, the effect of four putative PKC inhibitors {calphostin C, chelerythrine chloride, a pseudosubstrate
inhibitor for PKC [PKC peptide-(19
31)], and
staurosporine} on Ca2+
sensitization induced by acetylcholine (ACh) plus GTP was determined. Preincubation with each of the inhibitors did not affect subsequent Ca2+ sensitization induced by
muscarinic receptor stimulation in the presence of a constant
submaximal
[Ca2+]i,
neither did any of these compounds reverse the increase in Ca2+ sensitivity induced by ACh
plus GTP. Administration of a 1,2-diacylglycerol analog,
1-oleoyl-2-acetyl-sn-glycerol, did not induce
Ca2+ sensitization at a constant
submaximal
[Ca2+]i.
Thus we found no evidence that PKC mediates increases in
Ca2+ sensitivity produced by
muscarinic receptor stimulation in permeabilized CTSM.
-escin; calcium sensitivity; lung; trachea; canine; protein
kinase C inhibitors; activator; second messenger systems
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INTRODUCTION |
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IN AIRWAY SMOOTH MUSCLE, contraction is mediated by an increase in the concentration of cytosolic Ca2+ ([Ca2+]i). Ca2+ binds to calmodulin and subsequently increases myosin light chain kinase (MLCK) activity and phosphorylation of the 20-kDa regulatory myosin light chain (rMLC; see Ref. 8). rMLC phosphorylation allows the binding of myosin to actin, which increases actomyosin adenosinetriphosphatase activity and causes smooth muscle contraction. However, contractile force is not determined by [Ca2+]i alone (19), as membrane receptor stimulation with various agonists increases force developed at a constant submaximal [Ca2+]i, i.e., the Ca2+ sensitivity (1, 19).
In vascular (14) and colonic (24) smooth muscle, evidence exists that
protein kinase C (PKC) may act as a second messenger mediating
increases in Ca2+ sensitivity. In
tracheal smooth muscle, muscarinic receptor stimulation causes
translocation of PKC from the cytosol to the membrane, where it is
thought to be active (30). Both the putative PKC agonists phorbol
esters and muscarinic receptor stimulation increase Ca2+ sensitivity in -escin
permeabilized airway smooth muscle preparations (1, 3). However, other
studies in a variety of smooth muscles have not demonstrated an
involvement of PKC in the signal transduction pathway during membrane
receptor stimulation (7, 11, 23). The physiological role of PKC in
mediating membrane receptor agonist-induced increases in
Ca2+ sensitivity in airway smooth
muscle is unknown.
The purpose of the current study was to determine the role of PKC in
the regulation of Ca2+ sensitivity
in canine tracheal smooth muscle (CTSM) during muscarinic receptor
stimulation. We used a -escin permeabilized CTSM preparation in
which pores are produced in the cell membrane. In this way, [Ca2+]i
can be controlled by manipulating the extracellular
Ca2+ concentration, yet the
membrane-associated second messengers, including PKC, remain intact and
can be investigated (1, 3). We studied the effect of four putative PKC
inhibitors, calphostin C, chelerythrine chloride, a PKC pseudosubstrate
inhibitor [PKC peptide-(19
31) (PSSI)], and staurosporine
on Ca2+ sensitization produced by
acetylcholine (ACh) in the presence of GTP. We also examined the
ability of a specific PKC activator, the 1,2-diacylglycerol (DAG)
analog 1-oleoyl-2-acetyl-sn-glycerol (OAG), to increase
Ca2+ sensitivity at a constant
submaximal
[Ca2+]i.
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MATERIALS AND METHODS |
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Experimental Techniques
After Institutional Animal Care and Use Committee approval, and in conformance with the Guiding Principles for Research Involving Animals as approved by the Council of the American Physiological Society, mongrel dogs (n = 30; 15-23 kg) of either sex were anesthetized with an intravenous injection of pentobarbital sodium (30 mg/kg) and exsanguinated. The trachea was excised and immersed in chilled physiological salt solution (PSS) 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 under microscopic observation.Permeabilized CTSM. Muscle strips
(width 0.1-0.2 mm, length 3-5 mm, wet weight 0.05-0.1
mg) were mounted in 0.1-ml cuvettes and were continuously superfused at
2 ml/min with PSS (37°C) aerated with 94%
O2 and 6%
CO2, providing a pH of 7.4,
PO2 of
400 mmHg, and
PCO2 of
39 mmHg in the PSS. One
end of the strips 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). Optimal length
(Lo) of each
strip was achieved as previously described in detail (1, 3). The
remainder of the experiment was performed at 25°C.
The strips were permeabilized with -escin (18), a method validated
for CTSM in our laboratory (1).
-Escin creates pores in the cell
membrane, thus allowing substances of small molecular weight, such as
Ca2+, to freely diffuse across the
cell membrane. Accordingly,
[Ca2+]i
can be manipulated by changing the extracellular
Ca2+ concentration with ethylene
glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA)-buffered solutions in the bathing media. Larger cellular proteins necessary for contraction as well as the membrane
receptor-coupled second messenger systems are preserved after this
permeabilization procedure (1).
Muscle strips were permeabilized for 20 min with 100 µM -escin in
relaxing solution. The composition of the relaxing solution was as
follows: 7.5 mM ATP disodium salt, 4 mM EGTA, 20 mM imidazole, 1 mM
dithiothreitol, 1 nM free Ca2+, 10 mM creatinine phosphate, and 0.1 mg/ml creatinine phosphokinase. The pH
was buffered to 7.0 at 25°C with KOH; ionic strength was kept
constant at 0.20 M by adjusting the concentration of potassium acetate.
After permeabilization, strips were superfused with relaxing solution
for 10 min to wash out the excess
-escin. The
Ca2+ ionophore A-23187 (10 µM)
was added to the relaxing solution and all subsequent experimental
solutions to deplete the sarcoplasmatic reticulum
Ca2+ stores (16). Then, strips
were maximally contracted with 10 µM free
Ca2+; all subsequent isometric
force measurements were normalized to these contractions. After this
determination, strips were again superfused with relaxing solution
including Pi (5 mM) for 10 min to
reduce the time required for relaxation by accelerating the rate of
cross bridge detachment. To remove the
Pi, strips were superfused with
relaxing solution for 10 min, and the experimental protocol was
started. Solutions of varying free
Ca2+ concentrations were prepared
as previously described (1).
Intact CTSM. To determine the effects
of the PKC inhibitors in intact CTSM, muscle strips (width 0.5-1
mm, length 1.0-1.5 cm, weight 0.8-3 mg) were suspended in
25-ml water-jacketed tissue baths filled with PSS (37°C). PSS was
aerated with 94% O2 and 6%
CO2, providing a pH of 7.4,
PO2 of
400 mmHg, and PCO2 of
39 mmHg in the PSS. One
end of the strips was anchored to a metal hook at the bottom of the
tissue bath; the other end was attached to a calibrated force
transducer (model FT03D; Grass Instruments, Quincy, MA). During a 1-h
equilibration period, the strips were repeatedly contracted
isometrically for 30 s every 5 min by supramaximal electrical field
stimulation (EFS; 400 mA, 15 V, 25 Hz, 0.5-ms pulse duration). EFS was
triggered by a stimulator (model S88D; Grass Instruments) and was
delivered by a direct current amplifier (Section of Engineering, Mayo
Foundation). The length of the strips was increased after each
stimulation until
Lo was achieved.
Each strip was maintained at
Lo for the rest
of the experiment. During this equilibration period, the strips were
washed with fresh PSS every 10 min.
Experimental Protocols
Effect of PKC inhibitors on ACh-induced Ca2+ sensitization in permeabilized CTSM. We determined whether any of the four PKC inhibitors could prevent or reverse the increase in Ca2+ sensitivity induced by muscarinic receptor stimulation. These experiments included four tissue sets, one for each inhibitor. For each experiment, two permeabilized CTSM strips were prepared and studied concomitantly.
The two strips were superfused with relaxing solution alone (control) or in the presence (preincubation) of either 1 µM calphostin C (n = 5), 40 µM chelerythrine chloride (n = 5), 3 µM PSSI (n = 5), or 0.1 µM staurosporine (n = 5) for 10 min. Both strips were contracted with 0.3 µM free Ca2+ (equivalent to the free Ca2+ concentration producing 10% of isometric force in this preparation; see Ref. 3), one in the absence (control) and one in the presence (preincubation) of the PKC inhibitor for 10 min, the time required for stable contractile response. Then, both strips were contracted with 3 µM ACh plus 10 µM GTP for an additional 10 min, and force responses were measured. As previously determined (1), this ACh concentration produces 80% of its maximal effect on isometric force development at a constant [Ca2+]i in this preparation. To determine whether any of the four PKC inhibitors could reverse the increase in Ca2+ sensitivity induced by muscarinic receptor stimulation, the control CTSM strip was exposed to either 1 µM calphostin C (n = 5), 40 µM chelerythrine chloride (n = 5), 3 µM PSSI (n = 5), or 0.1 µM staurosporine (n = 5) for 10 min after contraction induced by 0.3 µM free Ca2+ containing 3 µM ACh plus 10 µM GTP had stabilized. The isometric force in the presence of the PKC inhibitor was measured and was compared with the value immediately before the addition of inhibitor. At the concentrations of free Ca2+ and ACh plus GTP chosen, contraction remains stable for the entire time course of the experimental protocol (unpublished observation).Effect of OAG on Ca2+ sensitivity in permeabilized CTSM. This experimental protocol was conducted to determine if OAG, a direct PKC activator, increases isometric force at a constant submaximal [Ca2+]i in a permeabilized smooth muscle preparation. Strips were contracted with 0.3 µM (n = 2) or 0.6 µM (n = 2) free Ca2+ for 10 min. Then, 100 µM OAG was added to the experimental solutions, and isometric force was continuously recorded for an additional 10 min. Subsequently, strips were stimulated with 0.3 µM free Ca2+ containing 3 µM ACh plus 10 µM GTP to demonstrate that the second messenger pathways mediating Ca2+ sensitivity were intact.
Effect of PKC inhibitors on ACh-induced contraction in intact CTSM. To determine if calphostin C, chelerythrine chloride, and staurosporine have any effects on CTSM, we performed additional experiments investigating their effect on intact tissues. Because the synthetic peptide PSSI cannot penetrate intact cells due to its polarity and size, it was not investigated in this protocol. Three muscle strips obtained from a single dog (n = 6) were studied to determine concentration-response curves to cumulative concentrations of staurosporine, chelerythrine chloride, and calphostin C. After each muscle strip was set at Lo, force was induced by 0.1 µM ACh (equivalent to the ACh concentration producing 50% of its maximal effect in isometric force development in intact CTSM) for 20 min, the time required for stable contractile response. The force developed was defined as the initial force response, and subsequent contractions recorded were normalized to this initial response (%initial force). Then, one muscle strip was exposed to 0 (control), 0.03, 0.1, 0.3, 1.0, 3.0, 6.0, and 10.0 µM staurosporine, the second strip was exposed to 0 (control), 1.0, 3.0, 6.0, 10.0, and 30.0 µM chelerythrine chloride, and the third strip was exposed to 0 (control), 0.1, 1.0, 10.0, and 100.0 µM and 1 mM calphostin C. For each concentration studied, stable relaxation was achieved in ~4 min. Additional strips were loaded with the Ca2+ fluorescent probe fura 2 acetoxymethyl ester (AM; n = 4) to investigate the effect of PKC inhibitors on [Ca2+]i in intact tissue. The emission fluorescence intensities due to excitation at 340 nm (F340) and 380 nm (F380) were measured, and the F340-to-F380 ratio was used as an index of [Ca2+]i. The effect of 0.1-3 µM staurosporine added to the perfusate on both F340/F380 and isometric force was studied. Due to the fluorescence properties of chelerythrine chloride and calphostin C, fura 2-AM fluorescence measurements could not be performed in our photometric system using these compounds. Materials. Fura 2-AM was purchased from Molecular Probes (Eugene, OR). ATP disodium salt was purchased from Research Organics (Cleveland, OH). PSSI was purchased from Boehringer Mannheim Biochemica (Indianapolis, IN). All other drugs and chemicals were purchased from Sigma Chemical (St. Louis, MO). A-23187, fura 2-AM, calphostin C, chelerythrine chloride, and staurosporine were dissolved in dimethyl sulfoxide (DMSO); in all experimental solutions, the final concentration of DMSO did not exceed 0.1%. At this concentration, DMSO had no effect on isometric force (data not shown). All other drugs and chemicals were prepared in distilled water. Statistical analysis. Data are expressed as mean values ± SD; n represents the number of dogs. To test for differences in the experimental groups, isometric force measurements were compared by paired Student's t-test. A P value ![]() |
RESULTS |
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Effect of PKC Inhibitors on ACh-Induced Ca2+ Sensitization in Permeabilized CTSM
Table 1 shows the effect of preincubation with 1 µM calphostin C, 40 µM chelerythrine chloride, 3 µM PSSI, and 0.1 µM staurosporine on isometric force induced by 0.3 µM free Ca2+ alone or by 0.3 µM free Ca2+ containing 3 µM ACh plus 10 µM GTP. Staurosporine in concentrations higher than 0.1 µM were not studied, since it attenuated force induced by 0.3 µM free Ca2+ alone (data not shown). This finding indicates that staurosporine at these concentrations has nonspecific effects other than inhibiting PKC. None of the PKC inhibitors significantly affected isometric force development induced by 0.3 µM free Ca2+ alone, nor did they prevent or attenuate Ca2+ sensitization induced by muscarinic receptor stimulation.
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Table 2 shows the effect of each of the four PKC inhibitors when added to strips in which Ca2+ sensitization was induced by 3 µM ACh plus 10 µM GTP at a constant submaximal [Ca2+]i of 0.3 µM. None of the PKC inhibitors reversed the increase in Ca2+ sensitivity induced by muscarinic receptor stimulation (Fig. 1).
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Effect of OAG on Ca2+ Sensitivity in Permeabilized CTSM
Adding 100 µM OAG to the permeabilized CTSM strips precontracted with either 0.3 or 0.6 µM free Ca2+ did not increase isometric force, i.e., did not increase Ca2+ sensitivity at a constant submaximal [Ca2+]i (Fig. 2). The increase in isometric force subsequently induced by 0.3 µM free Ca2+ containing 3 µM ACh plus 10 µM GTP was equivalent to the magnitude observed in the control experiments at a constant submaximal [Ca2+]i of 0.3 µM and muscarinic receptor stimulation.
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Effect of PKC Inhibitors on ACh-Induced Contraction in Intact CTSM
Based on the lack of effect in the use of the PKC inhibitors in
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The effect of staurosporine on [Ca2+]i in intact CTSM was demonstrated in experiments measuring fura 2-AM fluorescence. ACh (0.1 µM) increased both F340/F380 and isometric force. Subsequent addition of staurosporine (0.1-3 µM) consistently caused an irreversible decrease in both F340/F380 and isometric force (Fig. 4).
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DISCUSSION |
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The major findings of this study are that, in -escin permeabilized
CTSM 1) preincubation with each of
the four PKC inhibitors studied did not inhibit the increase in
Ca2+ sensitivity induced by
muscarinic receptor stimulation at a constant submaximal
[Ca2+]i,
2) administration of each of the
four PKC inhibitors did not reverse the increase in
Ca2+ sensitivity at a constant
submaximal
[Ca2+]i,
and 3) direct stimulation of PKC
with a DAG analog, OAG, did not increase
Ca2+ sensitivity at a constant
submaximal
[Ca2+]i.
-Escin permeabilized smooth muscle preparations have been used as a
tool to investigate the mechanisms regulating
Ca2+ sensitivity (1, 3, 18). The
creation of pores in the plasma membrane permits control of the
[Ca2+]i
by changing the extracellular Ca2+
concentrations in the solution superfusing the smooth muscle while
preserving large cellular proteins such as calmodulin, MLCK, rMLC,
actin, and myosin. With the use of this method, coupling of membrane
receptors to second messenger systems mediating
Ca2+ sensitivity is retained and
can be investigated (1, 3).
In our CTSM preparation, muscarinic receptor stimulation increases Ca2+ sensitivity (1, 3). This increase in Ca2+ sensitivity requires GTP, is mimicked by guanosine 5'-O-(3-thiotriphosphate), and is blocked by guanosine 5'-O-(2-thiodiphosphate), providing evidence for the involvement of GTP-binding proteins in this signal transduction pathway (3). The increase in Ca2+ sensitivity during membrane receptor stimulation is produced by the inhibition of rMLC phosphatase and increased rMLC phosphorylation (16). In vascular (14, 21) and colonic (24) permeabilized smooth muscle preparations, it has been suggested that membrane receptor agonists acting via GTP-binding proteins increase Ca2+ sensitivity in part by activating PKC. After receptor binding, phospholipase C is activated via a GTP-dependent process, producing DAG, a physiological activator of PKC, from phoshatidylinositol bisphosphate and phosphatidylcholine. It is thought that, once activated, PKC may directly or indirectly inhibit the rMLC phosphatases, increasing rMLC phosphorylation and isometric force at a constant submaximal [Ca2+]i (16). Increases in Ca2+ sensitivity induced by PKC activation might also be mediated by pathways not associated with increases in rMLC phosphorylation (15).
A role of PKC in agonist-induced
Ca2+ sensitization is supported by
the observation that phorbol esters such as phorbol 12,13-dibutyrate (PDBu; see Ref. 9) increase Ca2+
sensitivity in permeabilized smooth muscle preparations (3, 7, 24).
Further evidence for the involvement of PKC in agonist-induced Ca2+ sensitization has been
provided using PKC antagonists to reverse (4, 5, 21) or inhibit (5, 21)
agonist-induced increases in Ca2+
sensitivity in vascular smooth muscle. For example, 100 nM
staurosporine relaxed endothelin-1 (ET-1)-induced increases in
isometric force at a constant submaximal
[Ca2+]i
in -toxin permeabilized vascular smooth muscle but had little effect
on contractions elicited by Ca2+
alone (21). Chelerythrine chloride completely blocked the increase in
isometric force in response to ET-1 stimulation but could not reverse
it when added during steady-state agonist-induced force. Nishimura et
al. (21) concluded that the regulatory events important for the
maintenance of agonist-induced
Ca2+ sensitization occur between
PKC activation and the interaction of actin and myosin.
However, there is controversy about the involvement of PKC in mediating
the agonist-induced Ca2+
sensitization in different types of smooth muscle (7, 11, 23). In a
study using
-toxin permeabilized guinea pig stomach smooth muscle,
calphostin C (1 µM) and another PKC inhibitor, K-252b, had no effect
on ACh-induced Ca2+ sensitization
(23). Similarly, in
-escin permeabilized guinea pig vas deferens
smooth muscle, PSSI did not affect
Ca2+ sensitization induced by
norepinephrine or
(7).
This is the first study to investigate the role of PKC in mediating
agonist-induced Ca2+ sensitization
in permeabilized airway smooth muscle. Two different classes of PKC
antagonists were examined, which should inhibit the activity of most of
the isoforms of PKC present in CTSM [the Ca2+-dependent isoforms I and
II and the Ca2+-independent
isoforms
,
,
, and
(6)]. Two domains within PKC are
targets for the action of pharmacological inhibitors, a catalytic
domain that binds ATP and a regulatory domain that controls kinase
activity via binding of second messengers (e.g., adenosine
3',5'-cyclic monophosphate, guanosine
3',5'-cyclic monophosphate, and DAG). The interpretation of
our results of course assumes that the appropriate pharmacological
inhibitors were examined in the appropriate concentrations. We utilized
the four inhibitors up to the highest concentrations reported (4, 21,
23), well above published half-maximal inhibitory concentration
(IC50) values. Staurosporine, a
microbial product of Streptomyces staurosporeus (IC50 for PKC = 2.7 nM; see Ref.
29), and chelerythrine chloride, a naturally occurring alkaloid
(IC50 for PKC = 660 nM; see Ref. 10), both act on the catalytic domain. This ATP-binding
site exhibits considerable homology with other serine- and
threonine-specific kinases and tyrosine-specific kinases (28) so that
compounds acting at this site are considered to be rather nonselective. Calphostin C, derived from Cladosporium
cladosporioides (IC50 for PKC = 50 nM; see Ref. 17) and the pseudosubstrate inhibitor PSSI (directed
toward a 19 amino acid consensus sequence for PKC, IC50 = 147 nM; see Ref. 12) both
act at the regulatory site and are more selective.
None of the PKC inhibitors studied prevented or reversed ACh plus
GTP-induced increases in Ca2+
sensitivity (Fig. 1). Thus we found no evidence that PKC mediates increases in Ca2+ sensitivity
during muscarinic receptor stimulation in permeabilized CTSM. It must
be acknowledged that an isoform of PKC insensitive to inhibition by the
compounds studied may mediate agonist-induced Ca2+ sensitivity. However, OAG, a
DAG analog, stimulates
Ca2+-activated,
phospholipid-dependent isoforms of PKC, such as I and
II, which
are saliently expressed in CTSM (6) directly through coupling to the
binding site for activator molecules (20). The lack of effect of OAG on
Ca2+ sensitivity argues against a
physiological role of PKC in agonist-induced Ca2+ sensitization.
The addition of the phorbol ester PDBu to unstimulated intact tracheal
smooth muscle does not induce significant contraction (Ref. 2;
unpublished observations). However, we found that PDBu increases
Ca2+ sensitivity in a
concentration-dependent manner in our -escin permeabilized CTSM
preparation (1, 3). Because PDBu increases isometric force produced at
a given submaximal
[Ca2+]i
in permeabilized CTSM, it would appear that activation of PKC can,
under these circumstances, increase
Ca2+ sensitivity (1, 3). In
agreement with reports of
-toxin permeabilized rabbit mesenteric
artery smooth muscle (21), isometric force induced by PDBu developed
only in the presence of
0.1 µM [Ca2+]i,
demonstrating that the PKC isoforms mediating
Ca2+ sensitivity appear to be
primarily Ca2+ dependent. This
Ca2+ requirement might explain why
PDBu does not induce contraction in intact tracheal smooth muscle if
PDBu does not also significantly increase
[Ca2+]i
in this tissue. The effects of PDBu should be interpreted with caution
because the phorbol esters may activate PKC by mechanisms unlike those
involved during agonist stimulation, e.g., by forming complexes with
PKC (9). In vascular smooth muscle, it has been postulated that there
are at least two different pathways for Ca2+ sensitization, a
PKC-dependent pathway that is activated by phorbol esters and a
PKC-independent pathway that is activated by receptor agonists (11);
similar mechanisms may be present in CTSM.
Taken together, these previous studies and the current results suggest
that the involvement of PKC in agonist-induced increases in
Ca2+ sensitivity depends on the
type of smooth muscle studied, and, perhaps, the type of membrane
receptor agonist stimulation. This may reflect the fact that many
isoforms of PKC are present in smooth muscle, and their distribution
varies with species and smooth muscle type. With the use of rat
mesenteric small arteries, it has been reported that different PKC
isoforms in the same tissue mediate the contractile response to
different agonists (22). Additionally, it has been shown that the
distribution of PKC isoforms in ocular smooth muscle is not only
species specific but that each PKC isoform might have a specific
physiological function in mediating the contractile response (13).
However, the physiological role of
each isoform remains unclear. In CTSM, it has been suggested that the
Ca2+-dependent isoforms PKC-I
and -
II regulate the phosphorylation of MLCK (26).
Given our negative results of PKC inhibitors on ACh-induced
Ca2+ sensitization, we were
compelled to investigate whether they had any effect on intact CTSM.
Both staurosporine and chelerythrine chloride caused
concentration-dependent relaxation in intact CTSM (Fig. 3). These
findings suggest that the lack of effect of these compounds in the
-escin permeabilized CTSM preparation is unlikely to be the result
of a technical error. In prior studies, we found that staurosporine
also relaxed KCl-induced contractions in a concentration-dependent
manner (data not shown), which suggests that this compound has effects
independent of second messenger systems activated by membrane receptor
stimulation. Consistent with our findings, staurosporine also inhibits
contractile responses to KCl in vascular smooth muscle (25). Measuring
fura 2-AM fluorescence, we demonstrated that the staurosporine-induced
relaxation was associated with a decrease of
[Ca2+]i
(Fig. 4). These findings imply that PKC may be primarily involved in
the regulation of
[Ca2+]i
in CTSM. Exploration of the mechanisms by which PKC affects [Ca2+]i
are beyond the scope of this study. However, in other types of smooth
muscle, evidence exists for a role of PKC in regulating ion channels
(27).
In summary, none of the four pharmacological PKC inhibitors prevented
or reversed ACh plus GTP-induced increases in
Ca2+ sensitivity in -escin
permeabilized CTSM. Additionally, OAG, a direct DAG activator, did not
increase Ca2+ sensitivity at a
constant submaximal
[Ca2+]i.
Thus we found no evidence that PKC mediates increases in
Ca2+ sensitivity during muscarinic
receptor stimulation in canine airway smooth muscle.
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ACKNOWLEDGEMENTS |
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We thank Kathy Street and Rosimar Torres-Lèon for expert technical assistance and Janet Beckman and Cathy Nelson for preparing the manuscript.
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FOOTNOTES |
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This study was supported in part by National Heart, Lung, and Blood Institute Research Grants HL-45532 and HL-54757.
Address for reprint requests: K. A. Jones, Mayo Clinic and Mayo Foundation, 200 First St. SW, Rochester, MN 55905.
Received 26 March 1997; accepted in final form 23 June 1997.
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