Departments of Anesthesiology and Physiology and Biophysics, Mayo Clinic and Foundation, Rochester, Minnesota 55905
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ABSTRACT |
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We studied in -escin-permeabilized canine tracheal smooth
muscle (CTSM) the effect of the protein kinase C (PKC) agonist phorbol
12,13-dibutyrate (PDBu) on isometric force at a constant submaximal
Ca2+ concentration (i.e., the
effect on Ca2+ sensitivity) and
regulatory myosin light-chain (rMLC) phosphorylation. PDBu
increased Ca2+
sensitivity, an increase associated with a concentration-dependent, sustained increase in rMLC phosphorylation. PDBu altered the
relationship between rMLC phosphorylation and isometric force such that
the increase in isometric force was less than that expected for the increase in rMLC phosphorylation observed. The effect of four PKC
inhibitors [calphostin C, chelerythrine chloride, a
pseudosubstrate inhibitor for PKC, PKC peptide-(19
31) (PSSI), and
staurosporine] on PDBu-induced
Ca2+ sensitization as well as the
effect of calphostin C and PSSI on rMLC phosphorylation were
determined. Whereas none of these compounds prevented or reversed the
PDBu-induced increase in Ca2+
sensitivity, the PDBu-induced increase in rMLC phosphorylation was
inhibited. We conclude that PDBu increases rMLC phosphorylation by
activation of PKC but that the associated PDBu-induced increases in
Ca2+ sensitivity are mediated by
mechanisms other than activation of PKC in permeabilized airway smooth
muscle.
-escin; lung; trachea; canine; protein kinase C inhibitors; activator; phorbol 12,13-dibutyrate; second messenger systems; protein
kinase C
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INTRODUCTION |
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IN AIRWAY SMOOTH MUSCLE, contraction in response to physiological agonists is associated with an increase in cytosolic Ca2+ concentration ([Ca2+]i). The binding of Ca2+ to calmodulin increases myosin light-chain kinase (MLCK) activity and phosphorylation of the 20-kDa regulatory myosin light chain (rMLC) at the serine-19 and threonine-18 residues. rMLC phosphorylation at these sites allows the binding of myosin to actin, which increases actomyosin Mg2+-ATPase activity. However, smooth muscle contraction is not only regulated by [Ca2+]i, since membrane receptor stimulation increases isometric force developed at a constant submaximal [Ca2+]i, i.e., increases Ca2+ sensitivity (25).
In addition to GTP-binding proteins (10, 21), there is evidence in vascular (18) and colonic (27) smooth muscle that protein kinase C (PKC) mediates agonist-induced increases in Ca2+ sensitivity. The role of PKC is controversial, with results depending on the species, the type of smooth muscle, and the membrane receptor agonist studied. Data obtained in permeabilized nonvascular smooth muscle preparations have failed to demonstrate an involvement of PKC in this signal transduction (10, 26). In permeabilized canine trachea smooth muscle (CTSM), both the phorbol ester phorbol 12,13-dibutyrate (PDBu), a PKC agonist, and the muscarinic receptor agonist acetylcholine (ACh) increase Ca2+ sensitivity (4). Of interest, in permeabilized CTSM, pharmacological PKC inhibitors do not attenuate ACh-induced increases in isometric force at a constant submaximal [Ca2+], indicating that PKC does not mediate ACh-induced increases in Ca2+ sensitivity (5).
The purpose of the current study was to elucidate the mechanism by
which PDBu increases Ca2+
sensitivity in airway smooth muscle. We used a -escin-permeabilized CTSM preparation in which the
[Ca2+] at the
contractile apparatus can be controlled by manipulating the
[Ca2+] in solutions
bathing the muscle, yet intracellular second messenger systems,
including PKC, remain intact (3). We studied the effect of PDBu and
four pharmacological PKC inhibitors, calphostin C, chelerythrine
chloride, a PKC pseudosubstrate inhibitor [PKC peptide-(19
31) (PSSI)], and staurosporine, on isometric force and rMLC
phosphorylation at constant submaximal
[Ca2+].
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MATERIALS AND METHODS |
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Experimental Techniques
Following Institutional Animal Care and Use Committee approval, and in conformance with National Institutes of Health "Guide for the Care and Use of Laboratory Animals" [Department of Health and Human Services Publication No. (NIH) 85-23, Revised 1985], we anesthetized mongrel dogs (15-20 kg) of either sex with an intravenous injection of pentobarbital sodium (30 mg/kg) and exsanguinated them. 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.Isometric force measurements. 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 continuously superfused at 2 ml/min with PSS (37°C) aerated with 94% O2-6% 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 via stainless steel microforceps to a calibrated force transducer (model KG4, Scientific Instruments, Heidelberg, Germany). Optimal length of each strip was achieved as previously described (3, 4).
Permeabilization procedure and normalization of the isometric force
measurements.
Muscle strips were permeabilized with 100 µM -escin for 20 min at
25°C in Ca2+-free relaxing
solution according to a previously described protocol (3). The
composition of the relaxing solution was as follows (3): 7.5 mM MgATP,
4 mM EGTA, 20 mM imidazole, 1 mM dithiothreitol (DTT), 1 mM free
Mg2+, 1 nM free
Ca2+, 10 mM creatinine phosphate,
and 0.1 mg/ml creatine phosphokinase. The pH was buffered to 7.0 at
25°C with KOH, and ionic strength was kept constant at 0.2 M by
adjusting the concentration of potassium acetate. All experimental
solutions contained the Ca2+
ionophore A-23187 (10 µM) to deplete sarcoplasmic reticulum
Ca2+ stores, and all experimental
protocols were performed at 25°C. Before the experimental protocols
were started, the maximal contractile response for each strip was
determined by exposure to 10 µM free Ca2+; all subsequent isometric
force measurements were normalized to this response.
rMLC phosphorylation measurements.
rMLC phosphorylation was measured in separate sets of permeabilized
CTSM strips (width, 0.1-0.2 mm; length, 10-15 mm; wet weight,
0.1-0.15 mg). After an equilibration period of 30 min in aerated
PSS at 25°C, the strips were incubated in Ca2+-free PSS
containing 2mM EGTA for 15 min. Then, intracellular Ca2+ stores were depleted by
exposing the strips to 100 µM ACh for 10 min. ACh was removed from
the bath by exchanging solutions repeatedly with
Ca2+-free PSS over 15 min before
tissues were permeabilized. After experimental interventions, muscle
strips were flash-frozen by rapid immersion in acetone containing 10%
trichloroacedic acid (TCA) and 10 mM DTT cooled to 80°C with
crushed dry ice. rMLC was extracted as described by Gunst et al. (15).
Proteins were separated by glycerol-urea polyacrylamide gel (10%
acrylamide-0.5% bis-acrylamide) electrophoresis. All gels were
subjected to preelectrophoresis for 1 h at 400 V (10°C) to remove
urea and to allow DTT and thioglycolate to enter the gels.
Electrophoresis was conducted for 1 h at 100 V and then for 17 h at 400 V (10°C). Proteins were transferred to nitrocellulose membrane (0.2 µm) for 4 h at 1.6 A (15°C). Nitrocellulose sheets were washed
two times with 10 mM Tris-buffered saline containing 5% (wt/vol) BSA
for 1 h before labeling rMLC with polyclonal affinity-purified rabbit
anti-rMLC antibody (15). The anti-rMLC antibody was detected with
125I-labeled protein A (DuPont,
Boston, MA). The unphosphorylated and phosphorylated bands of rMLC were
visualized by phosphorimage analysis (PhosphorImager, Molecular
Dynamics, Sunnyvale, CA), and fractional phosphorylation was quantified
by ImageQuaNT software (Molecular Dynamics). After local background
subtraction, fractional phosphorylation was calculated as the ratio of
the sum of mono- and diphosphorylated rMLC to total rMLC.
Experimental Protocols
Four different experimental protocols were conducted using tissue obtained from separate groups of animals.Time-dependent effect of PDBu on rMLC phosphorylation. This protocol was conducted to establish the time course of PDBu's effect on changes in rMLC phosphorylation at a constant submaximal [Ca2+]. In preliminary studies, we confirmed that increases in rMLC phosphorylation produced by solutions containing 0.3 µM free Ca2+ alone are stable over 25 min (data not shown). Nine permeabilized CTSM strips were prepared from each of five dogs. One strip was not stimulated and was flash-frozen in relaxing solution for baseline rMLC phosphorylation measurements. Two strips were exposed to 0.3 µM free Ca2+ alone for 10 or 25 min before freezing. The remaining six strips were first exposed to 0.3 µM free Ca2+ alone for 10 min and then to 0.3 µM free Ca2+ containing 1 µM PDBu and flash-frozen at the following time points: 0.5, 1, 2, 5, 10, and 15 min. Free Ca2+ at 0.3 µM produces ~10% of maximal isometric force induced by 10 µM free Ca2+ in this preparation, and 1 µM PDBu produces ~90% of its maximal effect on isometric force at a constant 0.3 µM free Ca2+ (4). The time course of PDBu's effect on rMLC phosphorylation was investigated to determine the appropriate time point with the highest rMLC phosphorylation values to freeze the stimulated muscle in subsequent studies.
Concentration-dependent effect of PDBu on rMLC phosphorylation.
This protocol determined the effect of various PDBu concentrations on
rMLC phosphorylation at 0.3 µM free
Ca2+. In -escin-permeabilized
CTSM strips, PDBu (0.001-3 µM) causes a concentration-dependent
increase in isometric force at 0.3 µM free
Ca2+ (4). Five permeabilized
strips were prepared from each of six animals. One strip was not
stimulated and was flash-frozen in relaxing solution for baseline rMLC
phosphorylation measurements. Another strip was exposed to 0.3 µM
free Ca2+ for 25 min before
freezing. The remaining three strips were first exposed to 0.3 µM
free Ca2+ alone for 10 min and
then to 0.3 µM free Ca2+
containing one of three PDBu concentrations (0.01, 1, or 10 µM) for
15 min before freezing.
Effect of PDBu on the relationship between [Ca2+], rMLC phosphorylation, and isometric force. This protocol was conducted to establish the relationship between rMLC phosphorylation and isometric force induced by various [Ca2+] in the presence and absence of PDBu. For isometric force measurements, four permeabilized CTSM strips were prepared from each of six dogs. Two strips were contracted with 0.6 µM free Ca2+, one in the absence and one in the presence of 1 µM PDBu; the other two strips were contracted with 1 or 10 µM free Ca2+ alone. Isometric force was recorded for 15 min. At the concentrations of free Ca2+ and PDBu chosen, contractions remain stable for the time course of the experimental protocol.
For rMLC phosphorylation measurements, nine permeabilized CTSM strips were prepared from each of six dogs. One strip was not stimulated and was flash-frozen in relaxing solution for baseline rMLC phosphorylation measurements. The remaining eight muscle strips were stimulated with varying free Ca2+ concentrations (0.3, 0.6, 1, or 10 µM), four in the presence and four in the absence of 1 µM PDBu for 15 min before freezing.Effect of PKC inhibitors on PDBu-induced increases in isometric force and rMLC phosphorylation at a constant submaximal [Ca2+]. This protocol investigated whether PKC inhibitors could attenuate the PDBu-induced increases in Ca2+ sensitivity and rMLC phosphorylation. For isometric force measurements, we investigated whether calphostin C, chelerythrine chloride, PSSI, or staurosporine could prevent or reverse PDBu-induced increases in Ca2+ sensitivity. Four sets of two permeabilized CTSM strips were prepared and studied concomitantly, one for each inhibitor from each of five dogs. The two strips were superfused with relaxing solution alone (control) or in the presence (preincubation) of either 1 µM calphostin C, 40 µM chelerythrine chloride, 3 µM PSSI, or 0.1 µM staurosporine for 15 min. Both strips were contracted with 0.3 µM free Ca2+, one in the absence (control) and one in the presence (preincubation) of the PKC inhibitor for 10 min. Then, both strips were contracted with 0.3 µM free Ca2+ containing 1 µM PDBu in the absence (control) or presence (preincubation) of the PKC inhibitors for an additional 15 min, and isometric force responses were measured. To determine whether any of the four PKC inhibitors could reverse the increase in Ca2+ sensitivity induced by PDBu, the control CTSM strip was exposed to either 1 µM calphostin C, 40 µM chelerythrine chloride, 3 µM PSSI, or 0.1 µM staurosporine 15 min after the addition of 1 µM PDBu to solutions containing 0.3 µM free Ca2+ (Fig. 1). The isometric force in the presence of the PKC inhibitor was measured and compared with the value immediately before the addition of each inhibitor.
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Materials
The polyclonal affinity-purified rabbit anti-rMLC 20-kDa antibody was a generous gift of Dr. Susan J. Gunst, Department of Physiology and Biophysics, Indiana University School of Medicine, Indianapolis, IN. ATP, disodium salt, was purchased from Research Organics (Cleveland, OH). TCA was purchased from Fisher Scientific (Fair Lawn, NJ). The PKC inhibitor PSSI was purchased from Boehringer Mannheim Biochemica (Indianapolis, IN). All other drugs and chemicals were purchased from Sigma Chemical (St. Louis, MO). A-23187, calphostin C, chelerythrine chloride, PDBu, and staurosporine were dissolved in 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 or rMLC phosphorylation induced by free Ca2+ alone (data not shown). All other drugs and chemicals were prepared in distilled water.Statistical Analysis
Data are expressed as means ± SE for a minimum of five experiments; n represents the number of dogs. For multiple comparisons, a one-way ANOVA was performed. Differences in rMLC phosphorylation levels and isometric force were evaluated by Student's t-test for paired and unpaired data as appropriate. To determine whether PDBu altered the level of rMLC phosphorylation required to produce a given amount of isometric force, rMLC phosphorylation was calculated for the amount of isometric force during stimulation with PDBu using a nonlinear polynomial regression of the measured control isometric force values induced by free Ca2+ alone. This interpolated rMLC phosphorylation value was then compared with the measured rMLC phosphorylation value during PDBu stimulation by paired Student's t-test. A P value ![]() |
RESULTS |
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Time-Dependent Effect of PDBu on rMLC Phosphorylation
Stimulation with 0.3 µM free Ca2+ alone for 10 min significantly increased rMLC phosphorylation above baseline (from 15.7 ± 2.0 to 22.1 ± 2.6%). This Ca2+-induced increase in rMLC phosphorylation remained stable over the time course of the protocol (10 min, 22.1 ± 2.6%; 25 min, 21.9 ± 2.0%). Compared with 0.3 µM free Ca2+ alone, activation with 1 µM PDBu significantly increased rMLC phosphorylation after 5 min of PDBu exposure (from 22.1 ± 2.6 to 31.1 ± 2.78%), an increase that was sustained over the time course of the experiment (Fig. 2).
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Concentration-Dependent Effect of PDBu on rMLC Phosphorylation
Stimulation with 0.3 µM free Ca2+ alone for 10 and 25 min increased rMLC phosphorylation above baseline (from 18.2 ± 2.3 to 23.8 ± 2.3 and 21.9 ± 1.8%, respectively). Activation with 0.3 µM free Ca2+ for 10 min followed by 0.3 µM free Ca2+ containing 1 or 10 µM PDBu for 15 min significantly increased rMLC phosphorylation above baseline values and rMLC phosphorylation values produced by 0.3 µM free Ca2+ alone (from 21.9 ± 1.8 to 31.4 ± 2.5 and 33.4 ± 3.4%, respectively). PDBu (0.01 µM) did not significantly increase rMLC phosphorylation values above rMLC phosphorylation values induced by 0.3 µM free Ca2+ alone (from 21.9 ± 1.8 to 25.1 ± 3.1%) (Fig. 3).
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Effect of PDBu on the Relationship Between [Ca2+], rMLC Phosphorylation, and Isometric Force
Ca2+ caused a significant, concentration-dependent increase in both isometric force (Table 1) and rMLC phosphorylation (Fig. 4). The addition of 1 µM PDBu to solutions containing 0.6 µM free Ca2+ significantly enhanced Ca2+ sensitivity, increasing the isometric force (Table 1). Likewise, 1 µM PDBu significantly increased rMLC phosphorylation above that produced by each of the four free Ca2+ concentrations (Fig. 4). There was no significant difference in the magnitude of the PDBu-induced increase in rMLC phosphorylation between the Ca2+ concentrations (Fig. 4).
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PDBu significantly altered the relationship between rMLC phosphorylation and isometric force. The increase in isometric force induced by 0.6 µM free Ca2+ containing 1 µM PDBu was 51.1 ± 5.4% of maximal isometric force, and the corresponding rMLC phosphorylation value measured was 39.1 ± 4.4%. On the basis of nonlinear interpolation of the isometric force induced by free Ca2+ alone, the corresponding increase in rMLC phosphorylation was significantly greater than expected (measured rMLC phosphorylation, 39.1 ± 4.4% vs. expected rMLC phosphorylation, 34.2 ± 3.6%). Thus PDBu increased both isometric force and rMLC phosphorylation at a constant [Ca2+], but the amount of isometric force at this level of rMLC phosphorylation was less than predicted based on the relationship between rMLC phosphorylation and isometric force obtained during stimulation with Ca2+ alone (Fig. 5).
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Effect of PKC Inhibitors on PDBu-Induced Increases in Isometric Force and rMLC Phosphorylation at a Constant Submaximal [Ca2+]
Table 2 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 1 µM PDBu. Staurosporine at concentrations >0.1 µM was not studied, since these concentrations attenuated isometric force induced by 0.3 µM free Ca2+ alone (data not shown). This finding indicates that staurosporine at high concentrations has nonspecific effects on isometric force other than inhibiting PKC. None of the PKC inhibitors significantly affected the increase in isometric force induced by 0.3 µM free Ca2+ alone, nor did they prevent or attenuate the increase in isometric force induced by PDBu at a constant submaximal [Ca2+] (Fig. 1).
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Table 3 shows the effect of each of the four PKC inhibitors (treatment) when added to strips in which Ca2+ sensitization was induced by 1 µM PDBu at 0.3 µM free Ca2+ (control). None of the PKC inhibitors reversed the PDBu-induced increase in Ca2+ sensitivity.
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Neither calphostin C nor PSSI had an effect on baseline rMLC phosphorylation (control, 11.1 ± 1.2%; calphostin C, 10.3 ± 0.6%; PSSI, 9.9 ± 1.0%) or rMLC phosphorylation induced by 0.6 µM free Ca2+ alone (control, 20.3 ± 3.2%; calphostin C, 18.8 ± 3.0%; PSSI, 20.4 ± 2.8%). Table 4 shows the effect of preincubation with calphostin C and PSSI on rMLC phosphorylation levels induced by 0.6 µM free Ca2+ and 1 µM PDBu. There was no significant difference between rMLC phosphorylation levels induced by 0.6 µM free Ca2+ alone and 0.6 µM free Ca2+ containing 1 µM PDBu in the presence of the PKC inhibitors, indicating that PSSI and calphostin C prevented the increase in rMLC phosphorylation induced by activation of PKC.
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DISCUSSION |
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The major findings of this study in -escin-permeabilized CTSM are as
follows: 1) stimulation with the
phorbol ester PDBu increases rMLC phosphorylation via activation of
PKC; 2) this increase in rMLC
phosphorylation does not mediate PDBu-induced increases in
Ca2+ sensitivity; and
3) the PDBu-induced increase in
Ca2+ sensitivity does not involve
the activation of PKC at a constant submaximal
[Ca2+].
In vascular (11, 18) and colonic (27) smooth muscle, PKC is thought to
in part mediate agonist-induced
Ca2+ sensitization. Stimulation
with membrane receptor agonists is proposed to result in
phosphorylation of the 20-kDa rMLC via a GTP-dependent activation of
phospholipase C. Phospholipase C hydrolyzes phosphatidylinositol
4,5-bisphosphate and phosphatidylcholine, producing inositol
1,4,5-trisphosphate and diacylglycerol (DAG). By binding to the
regulatory domain of PKC, DAG and its analogs, such as
1-oleoyl-2-acetyl-sn-glycerol (OAG)
(29), can activate conventional and novel PKC isoforms. Once activated,
PKC may directly or indirectly inhibit the rMLC phosphatases (23).
Inhibition of rMLC phosphatases that dephosphorylate rMLC is thought to
be the major mechanism of agonist-induced
Ca2+ sensitization in smooth
muscle. Phorbol esters such as PDBu activate PKC by binding to the
regulatory domain of PKC (29) and thus inhibiting rMLC phosphatases
(23), increasing rMLC phosphorylation and isometric force at a constant
submaximal
[Ca2+]i
(21). Further evidence for a role of PKC in
Ca2+ sensitization has been
provided by using various pharmacological PKC inhibitors to reverse or
prevent phorbol ester-induced contractions in intact (28) and
permeabilized vascular (11, 18) and permeabilized vas deferens (10)
smooth muscle preparations. However, the involvement of PKC in
mediating Ca2+ sensitivity is
controversial, depending on the species, the type of smooth muscle, and
the kind of agonist used. Stimulation of various smooth muscle types
with physiological agonists like ACh (26), norepinephrine (10),
prostaglandin F2 (16) as well
as stimulation with AlF
4 (10) is
not inhibited by downregulation of PKC or by a variety of PKC
inhibitors.
In airway smooth muscle, the role of PKC in mediating contraction is
also unclear. The PKC isoforms present in CTSM have been identified and
include the conventional
Ca2+-dependent isoenzymes I and
II, the novel Ca2+-independent
isoenzymes
,
, and
, and the atypical isoenzyme
(8). The
atypical PKC isoenzyme
is insensitive to stimulation with PDBu, and
its activation is independent of
Ca2+, DAG, and phospholipid (7).
During contraction of CTSM, an agonist-induced translocation of PKC
from the cytosol to membrane fractions has been described, implying a
functional change from the inactive to the active, membrane-bound form
of PKC (33). In intact CTSM, phorbol esters induce an increase in
isometric force only after stimulation with KCl (Ref. 15; unpublished observations). After stimulation, this PDBu-induced increase in isometric force is accompanied by an increase in rMLC phosphorylation (15). In permeabilized CTSM, PDBu increases
Ca2+ sensitivity in a
concentration-dependent manner (2, 3), requiring a
[Ca2+] greater than
0.1 µM (3). This Ca2+
requirement may account for the lack of PDBu's effect in resting intact CTSM. In previous work, we demonstrated in
-escin-permeabilized CTSM that, at a constant submaximal
[Ca2+], treatment with
four pharmacological PKC inhibitors did not prevent or reverse
Ca2+ sensitization induced by ACh
(5). Additionally, OAG did not induce
Ca2+ sensitization at a constant
submaximal [Ca2+] (5).
Thus we found no evidence that PKC plays a physiological role in
mediating Ca2+ sensitivity during
muscarinic receptor stimulation in permeabilized CTSM. Rather, the role
of PKC in CTSM may be primarily to modulate [Ca2+]i
in intact muscle (5). These results raise the question of how PDBu
increases Ca2+ sensitivity in
permeabilized CTSM, if PKC does not contribute to
Ca2+ sensitization produced by the
physiological membrane receptor agonist ACh.
In this study, stimulation with PDBu induced a concentration-dependent, sustained increase in rMLC phosphorylation at constant [Ca2+]. However, there are two lines of evidence that the observed increase in rMLC phosphorylation may not be responsible for the increase in isometric force at a constant [Ca2+]. First, based on phosphopeptide mapping and sequence analysis in bovine tracheal tissue, Kamm et al. (19) showed that PDBu-induced activation of PKC mainly results in rMLC phosphorylation at the serine-1, serine-2, and threonine-9 residues (19). In contrast, in vascular smooth muscle, PDBu-induced PKC activation phosphorylates rMLC at serine-19 and threonine-18 only (11). It is thought that phosphorylation of the rMLC at sites other than serine-19 and threonine-18 does not to contribute to contraction; in fact, it has been shown that the additional phosphorylation of rMLC by PKC at threonine-9 attenuates the increase in Mg2+-ATPase activity associated with MLCK phosphorylation at serine-19 (19). In addition, phosphorylation of bovine tracheal myosin by PKC does not affect actin sliding velocity in an in vitro motility assay when the myosin had been previously phosphorylated by MLCK at serine-19 (32). In the current study, rMLC phosphorylation measurements performed detect total amounts of unphosphorylated and phosphorylated rMLC; phosphopeptide mapping to determine the site of phosphorylation was not performed. However, based on these previous studies, we speculate that the observed disparity in isometric force and expected level of rMLC phosphorylation during activation with PDBu at a constant submaximal [Ca2+] (Fig. 5) reflects phosphorylation by PDBu of rMLC sites that do not contribute to increases in isometric force. In support of this hypothesis, Gerthoffer and co-workers (12, 13) described a PDBu-induced increase in rMLC phosphorylation under some conditions in intact CTSM without a corresponding increase in isometric force.
Second, by using the more specific PKC inhibitors calphostin C and PSSI, we demonstrated that the PDBu-induced increase in rMLC is indeed mediated by activation of PKC, yet these inhibitors did not affect PDBu-induced Ca2+ sensitization. To block the effect of PDBu on conventional and novel PKC isoforms (31) present in CTSM, two different classes of distinct pharmacological PKC inhibitors were utilized. There are two possible targets for PKC inhibitors within PKC: a catalytic domain exhibiting substantial homology with other serine- and threonine-specific kinases and tyrosine-specific kinases (30), and a regulatory domain functioning as the binding site for DAG and its analogs. Staurosporine and chelerythrine chloride, both acting on the catalytic domain, are considered to be fairly nonspecific. Furthermore, staurosporine in concentrations higher than 0.1 µM attenuated isometric force induced by free Ca2+ alone (data not shown). Calphostin C and the pseudosubstrate inhibitor PSSI (directed toward a 19-amino acid consensus sequence for PKC) are more selective and both act on the regulatory site (30). All four PKC inhibitors were used in considerable excess of published IC50 values (5); these concentrations are thought to specifically inhibit PKC activity (26), whereas higher concentrations also significantly attenuate other kinases, e.g., MLCK (6); we observed evidence of this behavior at high concentrations of staurosporine (data not shown).
Recently, mechanisms in addition to rMLC phosphorylation have been proposed to mediate the effect of phorbol esters in smooth muscle through activation of PKC. In vitro studies have shown that the actin-binding proteins caldesmon and calponin are PKC substrates in solution, and phorbol esters, via activation of PKC isoforms, suppress calponin- and caldesmon-mediated inhibition of actomyosin Mg2+-ATPase (1, 24). Both of these proteins can be phosphorylated during smooth muscle contraction (1, 14, 24), but the physiological significance of this phosphorylation and the role of PKC in this process are unclear. The lack of effect of the PKC inhibitors on PDBu-induced calcium sensitization in our study does not support a role for a PKC-mediated process involving these actin-binding proteins.
The findings of this study suggest that PDBu increases Ca2+ sensitivity in permeabilized CTSM by activating an intracellular signal transduction pathway other than PKC. The fact that calphostin C and PSSI blocked PDBu-induced increases in rMLC phosphorylation without affecting isometric force demonstrate that PDBu can, under these circumstances, increase Ca2+ sensitivity by a mechanism that does not involve activation of PKC. It can, however, not be excluded that activation of PKC with PDBu could have induced a small increase in serine-19 and threonine-18 rMLC phosphorylation in the presence of the PKC inhibitors, an increase that could not be detected with our methods (Table 4). Several intracellular targets for PDBu other than PKC have recently been described. Different classes of proteins containing a single homologous cysteine-rich motif such as n-chimaerin (2) and Unc-13 (20) are high-affinity phorbol ester receptors whose binding affinities are similar to that of PKC. Although their role in smooth muscle contraction is unknown, the COOH-terminal domain of chimaerins is thought to perform as a GTPase-activating protein for the small GTP-binding protein p21rac (20). Furthermore, it has been demonstrated in permeabilized vascular smooth muscle that PDBu can phosphorylate a still unidentified 25-kDa protein or protein subunit (9). The exploration of these novel mechanisms by which PDBu might increase Ca2+ sensitization is beyond the scope of the current study.
In summary, PDBu caused an increase in
Ca2+ sensitivity at a constant
submaximal [Ca2+] in
-escin-permeabilized CTSM that was accompanied by an increase in
rMLC phosphorylation. Although the increase in rMLC phosphorylation was
inhibited by calphostin C and PSSI, none of the pharmacological PKC
inhibitors prevented or reversed PDBu-induced increases in isometric
force. These results suggest that PDBu increases rMLC phosphorylation
via activation of PKC, but this increase in rMLC phosphorylation is not
the mechanism by which PDBu increases isometric force at a constant
submaximal [Ca2+] in
CTSM. Furthermore, our findings give evidence of an as yet unidentified
pathway of phorbol ester-induced
Ca2+ sensitization other than
activation of PKC and rMLC phosphorylation.
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ACKNOWLEDGEMENTS |
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We thank Dr. Susan J. Gunst, Department of Physiology and Biophysics, Indiana University School of Medicine, Indianapolis, IN, for generously providing us with polyclonal affinity-purified rabbit anti-rMLC 20-kDa antibody as well as Dr. Cheryl A. Conover, Research Chair, and Dr. James B. Lawrence, Endocrine Research Unit, Mayo Clinic, Rochester, MN, for the use of the PhosphorImager. Our special thanks to Kathy Street and Robert Lorenz for expert technical assistance.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-45532 and HL-54757 and Deutsche Forschungsgemeinschaft Research Training Grant Br 1621/1-1.
Address for reprint requests: K. A. Jones, Mayo Clinic and Foundation, 200 First St. SW, Rochester, MN 55905.
Received 18 August 1997; accepted in final form 22 January 1998.
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REFERENCES |
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---|
1.
Adam, L. P.,
M. T. Franklin,
G. J. Raff,
and
D. R. Hathaway.
Activation of mitogen-activated protein kinase in porcine carotid arteries.
Circ. Res.
76:
183-190,
1995
2.
Ahmed, S.,
J. Lee,
R. Kozma,
A. Best,
C. Monfries,
and
L. Lim.
A novel functional target for tumor-promoting phorbol esters and lysophosphatidic acid. The p21rac-GTPase activating protein n-chimaerin.
J. Biol. Chem.
268:
10709-10712,
1993
3.
Akao, M.,
A. Hirasaki,
K. A. Jones,
G. Y. Wong,
D. H. Bremerich,
and
D. O. Warner.
Halothane reduces myofilament Ca2+ sensitivity during muscarinic receptor stimulation of airway smooth muscle.
Am. J. Physiol.
271 (Lung Cell. Mol. Physiol. 15):
L719-L725,
1996
4.
Bremerich, D. H.,
A. Hirasaki,
K. A. Jones,
and
D. O. Warner.
Halothane attenuation of calcium sensitivity in airway smooth muscle.
Anesthesiology
87:
94-101,
1997[Medline].
5.
Bremerich, D. H.,
D. O. Warner,
R. R. Lorenz,
R. Shumway,
and
K. A. Jones.
Role of protein kinase C in calcium sensitization during muscarinic stimulation in airway smooth muscle.
Am. J. Physiol.
273 (Lung Cell. Mol. Physiol. 17):
L775-L781,
1997
6.
Brozovich, F. V.
PKC regulates agonist-induced force enhancement in single alpha-toxin-permeabilized vascular smooth muscle cells.
Am. J. Physiol.
268 (Cell Physiol. 37):
C1202-C1206,
1995
7.
Clément-Chomienne, O.,
and
M. P. Walsh.
Identification of protein kinase C isoenzymes in smooth muscle: partial purification and characterization of chicken gizzard PKC .
Biochem. Cell. Biol.
74:
51-65,
1996[Medline].
8.
Donnelly, R.,
K. Yang,
M. B. Omary,
S. Azhar,
and
J. L. Black.
Expression of multiple isoenzymes of protein kinase C in airway smooth muscle.
Am. J. Respir. Cell. Mol. Biol.
13:
253-256,
1995[Abstract].
9.
Foster, C. J.,
and
M. Chatterjee.
A 25,000 dalton protein is phosphorylated in response to phorbol ester stimulation in skinned carotid artery (Abstract).
Biophys. J.
49:
73a,
1986.
10.
Fujita, A.,
T. Takeuchi,
H. Nakajima,
H. Nishio,
and
F. Hata.
Involvement of heterotrimeric GTP-binding protein and rho protein, but not protein kinase C, in agonist-induced Ca2+ sensitization of skinned muscle of guinea pig vas deferens.
J. Pharmacol. Exp. Ther.
274:
555-561,
1995[Abstract].
11.
Gailly, P.,
M. C. Gong,
A. V. Somlyo,
and
A. P. Somlyo.
Possible role of atypical protein kinase C activated by arachidonic acid in Ca2+ sensitization of rabbit smooth muscle.
J. Physiol. (Lond.)
500:
95-109,
1997[Abstract].
12.
Gerthoffer, W. T.
Calcium dependence of myosin phosphorylation and airway smooth muscle contraction and relaxation.
Am. J. Physiol.
250 (Cell Physiol. 19):
C597-C604,
1986
13.
Gerthoffer, W. T.,
K. A. Murphey,
and
S. J. Gunst.
Aequorin luminescence, myosin phosphorylation, and active stress in tracheal smooth muscle.
Am. J. Physiol.
257 (Cell Physiol. 26):
C1062-C1068,
1989
14.
Gerthoffer, W. T.,
I. A. Yamboliev,
J. Pohl,
R. Haynes,
S. Dang,
and
J. McHugh.
Activation of MAP kinases in airway smooth muscle.
Am. J. Physiol.
272 (Lung Cell. Mol. Physiol. 16):
L244-L252,
1997
15.
Gunst, S. J.,
M. H. al-Hassani,
and
L. P. Adam.
Regulation of isotonic shortening velocity by second messengers in tracheal smooth muscle.
Am. J. Physiol.
266 (Cell Physiol. 35):
C684-C691,
1994
16.
Hori, M.,
K. Sato,
S. Miyamoto,
H. Ozaki,
and
H. Karaki.
Different pathways of calcium sensitization activated by receptor agonists and phorbol esters in vascular smooth muscle.
Br. J. Pharmacol.
110:
1527-1531,
1993[Abstract].
17.
House, C.,
and
B. E. Kemp.
Protein kinase C contains a pseudosubstrate prototope in its regulatory domain.
Science
238:
1726-1728,
1987[Medline].
18.
Itoh, T.,
A. Suzuki,
and
Y. Watanabe.
Effect of a peptide inhibitor of protein kinase C on G-protein-mediated increase in myofilament Ca2+-sensitivity in rabbit arterial skinned muscle.
Br. J. Pharmacol.
111:
311-317,
1994[Abstract].
19.
Kamm, K. E.,
L. C. Hsu,
Y. Kubota,
and
J. T. Stull.
Phosphorylation of smooth muscle myosin heavy and light chains. Effects of phorbol dibutyrate and agonists.
J. Biol. Chem.
264:
21223-21229,
1989
20.
Kazanietz, M. G.,
N. E. Lewin,
J. D. Bruns,
and
P. M. Blumberg.
Characterization of the cysteine-rich region of the Caenorhabditis elegans protein Unc-13 as a high affinity phorbol ester receptor. Analysis of ligand-binding interactions, lipid cofactor requirements, and inhibitor sensitivity.
J. Biol. Chem.
270:
10777-10783,
1995
21.
Kitazawa, T.,
B. D. Gaylinn,
G. H. Denney,
and
A. P. Somlyo.
G-protein-mediated Ca2+ sensitization of smooth muscle contraction through myosin light chain phosphorylation.
J. Biol. Chem.
266:
1708-1715,
1991
22.
Kobayashi, E.,
H. Nakano,
M. Morimoto,
and
T. Tamaoki.
Calphostin C (UCN-1028C), a novel microbial compound, is a highly potent and specific inhibitor of protein kinase C.
Biochem. Biophys. Res. Commun.
159:
548-553,
1989[Medline].
23.
Masuo, M.,
S. Reardon,
M. Ikebe,
and
T. Kitazawa.
A novel mechanism for the Ca2+-sensitizing effect of protein kinase C on vascular smooth muscle: inhibition of myosin light chain phosphatase.
J. Gen. Physiol.
104:
265-286,
1994[Abstract].
24.
Mino, T.,
U. Yuasa,
M. Naka,
and
T. Tanaka.
Phosphorylation of calponin mediated by protein kinase C in association with contraction in porcine coronary artery.
Biochem. Biophys. Res. Commun.
208:
397-404,
1995[Medline].
25.
Morgan, J. P.,
and
K. G. Morgan.
Stimulus-specific patterns of intracellular calcium levels in smooth muscle of ferret portal vein.
J. Physiol. (Lond.)
351:
155-167,
1984[Abstract].
26.
Oishi, K.,
M. Mita,
T. Ono,
T. Hashimoto,
and
M. K. Uchida.
Protein kinase C-independent sensitization of contractile proteins to Ca2+ in alpha-toxin-permeabilized smooth muscle cells from the guinea-pig stomach.
Br. J. Pharmacol.
107:
908-909,
1992[Abstract].
27.
Sato, K.,
R. Leposavic,
N. G. Publicover,
K. M. Sanders,
and
W. T. Gerthoffer.
Sensitization of the contractile system of canine colonic smooth muscle by agonists and phorbol ester.
J. Physiol. (Lond.)
481:
677-688,
1994[Abstract].
28.
Shimamoto, Y.,
H. Shimamoto,
C. Y. Kwan,
and
E. E. Daniel.
Differential effects of putative protein kinase C inhibitors on contraction of rat aortic smooth muscle.
Am. J. Physiol.
264 (Heart Circ. Physiol. 33):
H1300-H1306,
1993
29.
Slater, S. J.,
M. B. Kelly,
F. J. Taddeo,
E. Rubin,
and
C. D. Stubbs.
Evidence for discrete diacylglycerol and phorbol ester activator sites on protein kinase C. Differences in effects of 1-alkanol inhibition, activation by phosphatidylethanolamine and calcium chelation.
J. Biol. Chem.
269:
17160-17165,
1994
30.
Tamaoki, T.,
and
H. Nakano.
Potent and specific inhibitors of protein kinase C of microbial origin.
Biotechnology
8:
732-735,
1990[Medline].
31.
Thomson, F. J.,
M. S. Johnson,
R. Mitchell,
W. B. Wolbers,
A. J. Ison,
and
D. J. MacEwan.
The differential effects of protein kinase C activators and inhibitors on rat anterior pituitary hormone release.
Mol. Cell. Endocrinol.
94:
223-234,
1993[Medline].
32.
Umemoto, S.,
A. R. Bengur,
and
J. R. Sellers.
Effect of multiple phosphorylations of smooth muscle and cytoplasmic myosins on movement in an in vitro motility assay.
J. Biol. Chem.
264:
1431-1436,
1988
33.
Yamakage, M.
Direct inhibitory mechanisms of halothane on canine tracheal smooth muscle contraction.
Anesthesiology
77:
546-553,
1992[Medline].