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Department of Medicine, Rhode Island Hospital and Brown Medical School, Providence, Rhode Island 02903 (K.M.H., J.B., P.B.), Department of Pharmacology, Yeungnam Medical School, Taegu 705-035, Korea (U.D.S.), Schepens Eye Research Institute, Boston, Massachusetts 02114 (D.Z., D.D.), and Institut Pasteur de Lille, Lille, France (C.S.)
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Summary |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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Circular muscle of the esophagus (ESO) is normally relaxed and
contracts phasically in response to neural stimuli. In contrast, lower
esophageal sphincter (LES) circular muscle maintains spontaneous tone
and relaxes in response to neural stimuli. We have previously shown
that in vitro, spontaneous LES tone and contraction of
ESO in response to acetylcholine (ACh) are antagonized by protein kinase C (PKC) inhibitors, suggesting that PKC activation is
responsible for these functions. In the current study, Western blot
analysis of LES and ESO revealed PKC-
, -
II, and -
isozymes in
LES circular muscle, but only PKC-II translocated from the cytosolic
to the membrane fraction in response to ACh. In contrast, ESO contained PKC-II, -, and -
, and only PKC- translocated to the
membrane fraction in response to ACh. In LES single cells isolated by
enzymatic digestion and permeabilized by saponin,
1-2-dioctanoylglycerol-mediated contraction was inhibited by
preincubation with PKC-II antiserum but not by other PKC antisera.
In esophageal cells, contraction was inhibited by the PKC- antiserum
but not by antisera against other PKC isozymes.
N-Myristoylated peptides derived from the pseudosubstrate sequences of PKC isozymes were used to inhibit saponin,
1-2-dioctanoylglycerol-induced contraction of LES and ESO smooth
muscle cells. Contraction of LES cells was reduced by the
pseudosubstrate but not by the , 
, or pseudosubstrate. Contraction of ESO cells was reduced by the pseudosubstrate but not
by the , , or pseudosubstrate. We conclude that different types of contractile activity in the ESO and LES are mediated
by different PKC isozymes. LES contraction is mediated by the
calcium-dependent PKC-II, whereas contraction of ESO is mediated by
the calcium-independent PKC-.
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Introduction |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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ESO circular smooth muscle is normally relaxed and contracts with a brief and powerful "phasic" contraction in response to neural (cholinergic) stimuli induced by swallowing. LES circular muscle maintains spontaneous myogenic tone and relaxes in response to neural (nonadrenergic-noncholinergic inhibitory) stimuli.
We have previously shown that both ESO contraction in response to a maximally effective dose of ACh and LES tone are mediated by PKC-dependent intracellular transduction pathways. ACh-induced contraction of ESO circular smooth muscle requires the influx of extracellular calcium and activation of PKC and is independent of calmodulin (1, 2). In addition, the calcium influx is required primarily for the activation of the phospholipases responsible for the production of DAG and not for the direct activation of PKC (2). In contrast, LES tone is mediated by a calcium-dependent PKC pathway. During the maintenance of tone, spontaneous phospholipase C activity produces low levels of Ins(1,4,5)P3 and DAG. Ins(1,4,5)P3 causes the release of low levels of calcium from intracellular stores (3). However, different PKC isozymes may regulate these two contractile processes in these distinct tissue types.
PKC is a family of homologous serine and threonine protein kinases.
Eleven isozymes of PKC have been identified in mammalian tissues. These
isozymes can be divided into three groups based on their calcium and
phospholipid requirements for activation: classic, or conventional PKC
(cPKC), including , I, II, and , which are calcium and
phospholipid dependent; new PKC (nPKC), including , , 
, 
,
and µ, which are calcium independent and phospholipid dependent; and
atypical PKC (aPKC), including 
and 
, which are calcium and
phospholipid independent (4).
In the current study, we examined the PKC isozymes present in the ESO
and LES and investigated which ones mediate LES basal tone and
ACh-induced contraction of ESO. We found that LES contraction is
mediated by the calcium-dependent PKC- isozymes, whereas contraction of the ESO is mediated by the calcium-independent PKC-.
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Materials and Methods |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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Tissue dissection and dispersion of smooth muscle cells. Adult cats of either sex weighing 3-5 kg were used, and ESO and LES smooth muscle squares were prepared as previously described (1, 5). The chest and abdomen were opened with a midline incision exposing the ESO and stomach. The ESO and stomach were removed together and pinned onto a wax block at their in vivo dimensions and orientation. The ESO and stomach were opened along the lesser curvature. The location of the squamocolumnar junction was identified, and the mucosa was peeled. The high pressure zone is characterized by a visible thickening of the circular muscle layer in correspondence of the squamocolumnar junction and immediately proximal to the sling fibers of the stomach. We have previously shown that a 5-8-mm band of tissue that coincides with the thickened area constitutes the LES and has distinct characteristics when examined in vivo, in the organ bath, or as single cells after enzymatic digestion (1, 6).
After the ESO and stomach were opened and the LES was identified as described above, the mucosa and submucosal connective tissue were removed by sharp dissection. The LES was excised, and a 3-5-mm-wide strip at the junction of LES and ESO was discarded to avoid overlap. The circular muscle layer from LES and ESO was cut into 0.5-mm-thick slices with a Stadie Riggs tissue slicer (Thomas Scientific Apparatus, Philadelphia, PA). The last slices, containing the myenteric plexus, longitudinal muscle, and serosa, were discarded; then, the slices were cut by hand into 2 × 2-mm tissue squares. Tissue squares of circular muscle were used either in Western blot analysis or further digested to isolated single cells for contractility studies. Tissue squares were digested in HEPES buffer containing 0.1% collagenase type II to isolate smooth muscle cells as previously described (1); the HEPES solution contained 115 mM NaCl, 5.8 mM KCl, 12 mM KH2PO4, 2.5 mM glucose, 25 mM HEPES, 2 mM CaCl2, 0.6 mM MgCl2, 0.3 mg/ml BME amino acid supplement, and 0.09 mg/ml soybean trypsin inhibitor. The solution was gently gassed with 100% O2. At the end of the digestion period, the tissue was poured over a 450-µm nylon mesh (Tetko, Elmsford, NY), rinsed in collagenase-free HEPES buffer to remove any trace of collagenase, and then incubated in this solution at 31° and gassed with 100% O2. The cells were allowed to dissociate freely for 10-20 min. Cells were permeabilized, when required, to control intracellular calcium concentration and to allow the use of agents such as PKC antibodies, which do not diffuse across the intact cell membrane. After completion of the enzymatic phase of the digestion process, the partly digested muscle tissue was washed with an enzyme-free cytosolic buffer composed of 20 mM NaCl, 100 mM KCl, 5.0 mM MgSO4, 0.96 mM NaH2PO4, 1.0 mM EGTA, 0.48 mM CaCl2, and 2% bovine serum albumin. The cytosolic buffer was equilibrated with 95% O2/5% CO2 to maintain pH 7.2 at 31°. Muscle cells dispersed spontaneously in this medium. After dispersion, the cells were permeabilized by incubation for 3 min in cytosolic buffer containing 75 µg/ml saponin. After exposure to saponin, the cell suspension was spun at 500 × g, and the resulting pellet was resuspended in saponin-free modified cytosolic buffer containing 10 µM antimycin A, 1.5 mM ATP, and an ATP-regenerating system consisting of 5 mM creatine phosphate and 10 units/ml creatine phosphokinase. After the cells were washed free of saponin, they were resuspended in modified cytosolic buffer.Agonist-induced contraction of isolated muscle cells.
The
cells were contracted by exposure for 30 sec to DAG (10
7
M for ESO and 106 M for LES) in
permeabilized cells. These concentrations produce a maximal contraction
of the circular smooth muscle from these two distinct tissue types.
7 M peptide for 60 min
before the addition of DAG. We have previously shown that preincubation
of lacrimal gland acini for 60 min with 107 M
myr-PKC- results in 80% inhibition of protein secretion by phorbol
esters (9). A myristoylated peptide derived from the sequence of the
endogenous inhibitor of cAMP-dependent protein kinase A, indicated in
the figure as PKI, was used as a negative control to test the sequence
specificity of the PKC pseudosubstrate peptides.
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Polyacrylamide gel electrophoresis and immunoblotting.
The
identification of PKC isozymes in LES and ESO circular muscle was
performed by Western blot analysis. Briefly, tissue squares of LES and
ESO circular muscle were homogenized in phosphate buffer and
centrifuged for 1 min at low rpm. The supernatant was mixed with SDS
buffer, boiled for 5 min, and centrifuged at 12,000 rpm for 5 min at
4°. Prestained molecular mass marker was prepared in the same manner.
After these supernatant samples were subjected to SDS-polyacrylamide
gel electrophoresis (10), the separated proteins were
electrophoretically transferred to an NC membrane (BioRad, Melville,
NY) at 60 V for 5 hr, followed by 30 V overnight. Transfer of proteins
to the NC membrane was confirmed with Ponseau S staining reagent. To
block nonspecific binding, the NC membrane was incubated in 5% nonfat
dry milk in phosphate-buffered saline for 60 min, followed by three
rinses in milk-free buffer. Incubation with 1:1000 dilution of antibody
raised against each PKC isozyme (i.e., , I, II, , , ,
) was done for 1 hr with shaking, followed by three washes with
antibody-free buffer. This was followed by a 60-min incubation in
horseradish peroxidase-conjugated goat anti-rabbit antibody. Detection
was achieved with an enhanced chemiluminescence agent. Molecular mass
was estimated by comparison of sample bands with a prestained molecular
mass marker.
7 M for LES
and 105 M for ESO). These concentrations of
ACh have previously been determined to produce maximal PKC-dependent
contraction in the two different tissue types. After 30 sec, the
reaction was stopped with 10 volumes of ice-cold Krebs' solution.
Tissue squares were collected and homogenized in 20 mM Tris
buffer, pH 7.4, containing 0.5 M EDTA, 0.5 M
EGTA, 10 mg/ml leupeptin, 10 mg/ml aprotinin, and 10 mM
-mercaptoethanol. Homogenization consisted of two 10-sec bursts with
a Tissue Tearer (Biospec, Racine, WI) followed by 40-60 strokes with a
Dounce tissue grinder (Wheaton, Melville, NJ). Samples were centrifuged
at 100,000 × g for 40 min at 4° (50 Ti rotor;
Beckman Ultracentrifuge, Palo Alto, CA). The supernatant was collected
and, after the addition of 30 µl of 10 mg/ml phenylmethylsulfonyl fluoride and a 30-min incubation, was used as the cytosolic fraction. The pellet was resuspended in 3 ml of homogenizing buffer containing 0.1% Triton X-100 and rehomogenized by 20 strokes with a Dounce tissue
grinder. The resuspended sample was mixed well by the use of a tube
rotator for 30-40 min at 4° and then centrifuged at 100,000 × g for 50 min at 4°. The supernatant of this centrifugation was collected as the membrane fraction. The identification of PKC
isozymes in the cytosolic and membrane fractions was performed by
Western blot analysis as described above. The bands were analyzed using
scanning densitometry (Howtek, Hudson, NH).
In addition to Western blot analysis, ACh-stimulated translocation of
PKC activity was measured by colorimetric assay. The cytosolic and
membrane fractions were immunoprecipitated by isozyme-specific PKC
antibodies, and PKC activity was measured. Briefly, 5 µg of isozyme-specific PKC antibodies (PKC-II for LES and PKC- for ESO)
were added to 400 µl of cytosolic or membrane fraction and incubated
for 1 hr at 4°. Then, 40 µl of protein A/G PLUS-agarose (Santa Cruz
Biochemicals, Santa Cruz, CA) was added, and samples were incubated at
4° with rocking. After 1 hr, samples were microcentrifuged (Fisher
Scientific, Pittsburgh, PA) for 15-20 sec at 4°. The pellet was
suspended in 20 µl of RIFA buffer (1 mM
KH2PO4, 10 mM
Na2HPO4, 137 mM NaCl, 2.7 mM KCl, 1% tergitol, 0.5% sodium deoxycholate, 0.1% SDS,
pH 7.4) and solubilized. The supernatant was removed, and the pellet
was suspended in 20 µl of RIFA buffer and solubilized. Ten
microliters of solubilized sample was used in the PKC colorimetric assay.
Measurement of PKC activity. PKC activity of immunoprecipitated smooth muscle of the LES and ESO was measured using the Pierce Colorimetric PKC Assay no. 29510 (Pierce, Rockford, IL). Briefly, a peptide substrate that was labeled with brightly colored fluorescent dye was incubated with the kinase-containing sample. The reaction mixture was applied to an affinity column that binds phosphorylated peptides. The phosphorylated product was eluted from the column and quantified by measurement of its absorbance at 570 nm.
Protein determination. Protein content was obtained after hydrolysis by 0.1 N NaOH at 80° to solubilize the protein, followed by neutralization with HC1. The amount of protein present was determined by colorimetric analysis (BioRad Protein Assay; BioRad, Richmond, CA) according to the method of Bradford (11).
Pseudosubstrate peptide synthesis. Myristoylated peptides were synthesized by BOC strategy on an MHBA resin (Novabiochem, Meudon, France) using an Applied Biosystems 430A automated synthesizer (Foster City, CA). Protocols and reagents were used as recommended by the manufacturer. Myristic acid was coupled to the peptide using dicyclohexylcarbdiimide hydroxybenzotriazole. Peptides were purified by reverse-phase high performance liquid chromatography on a Vydac C4 (30 × 0.9-cm) preparative column using a trifluoroacetic acid/acetonitrile solvent system. Peptide integrity was monitored by amino acid analysis and mass spectrometry.
Drugs and chemicals.
PKC antibodies (i.e., , I, II,
, , , ) were from GIBCO BRL (Gaithersburg, MD). Enhanced
chemiluminescence agents and rainbow-prestained molecular mass markers
were from Amersham (Arlington Heights, IL). Horseradish
peroxidase-conjugated goat anti-rabbit antibody was from Pierce.
Collagenase type II and soybean trypsin inhibitor were from Worthington
Biochemicals (Freehold, NJ). SDS sample buffer was from BioRad
(Hercules, CA ). Saponin, Ponseau S, BME amino acid supplement, EGTA,
HEPES, creatine phosphate, creatine phosphokinase, ATP, antimycin A,
and other reagents were purchased from Sigma Chemical (St. Louis, MO).
Statistical analysis. Estimates of the half-maximal response to the myristoylated pseudosubstrate peptides were determined by interpolation from graphs of log concentration versus logit values of percent shortening (12). Data are expressed as mean ± standard error. Statistical differences between groups were determined by Student's t test. Differences between multiple groups were tested using ANOVA for repeated measures and checked for significance using Scheffé's F test.
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Results |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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Western blot analysis of PKC isozymes.
Fig. 1
shows that PKC isozymes, detected by Western blot analysis in the
circular layer of LES smooth muscle, include the calcium-dependent
PKC-, -II, and - isozymes. In contrast, the PKC-II, -,
and - isozymes are present in the circular smooth muscle of the ESO.
The specificity of each immunoreactive band was substantiated by the
blockade of the signal after incubation of the corresponding peptide
antigen together with the antibody directed against each PKC isozyme
(Fig. 1, lane 1).
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Different PKC isozymes translocate in response to ACh in LES and ESO. Activation of PKC is associated with the translocation of the enzyme from the cytosol to the membrane fraction (13, 14). To test the functional significance of the PKC isozymes present in LES and ESO circular smooth muscle, we examined their translocation from cytosol to membrane in response to ACh stimulation.
Western blot analysis revealed that among the PKC isozymes present in LES circular smooth muscle, only PKC-II translocates from the
cytosol (Fig. 2, lane 1) to the membrane
(Fig. 2, lane 4) in response to ACh stimulation. In ESO
circular smooth muscle, only PKC- translocates from the cytosol to
the membrane in response to ACh.
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II bands was present in
the cytosolic fraction, whereas the remaining 28% was found in the
membrane. After ACh stimulation, these proportions reverse, with 70%
of total PKC-II band density present in the membrane fraction and
30% density remaining in the cytosol. In the ESO under control
conditions, 72% of the total density of the PKC- bands was present
in the cytosolic fraction and 28% was in the membrane fraction. After
ACh stimulation, 68% of total PKC- band density was present in the
membrane fraction, with 30% density remaining in the cytosol. Fig. 3B
demonstrates that the ratio of density (membrane/cytosol) of PKC-II
in the LES and PKC- in the ESO significantly increases after ACh
stimulation (p < 0.01, paired t
test).
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II for LES and for ESO), and PKC activity was
measured by colorimetric assay. The ratios of PKC activity
(membrane/cytosol) of the II isozyme for the LES and the isozyme
for the ESO were significantly increased after ACh stimulation
(p < 0.01, paired t test).
Inhibition of DAG-induced contraction of permeabilized LES and ESO cells by PKC antibodies. To confirm that PKC-mediated contraction may be isozyme specific, we examined the effect of different PKC antibodies on contraction induced by the endogenous PKC activator DAG .
Fig. 4A shows that DAG-induced contraction of permeabilized LES cells was significantly inhibited by antibodies raised against PKC-II and not by antibodies raised against the
PKC-, -, or - isozyme (ANOVA, p < 0.01).
Inhibition of DAG-induced contraction PKC-II was concentration
dependent (Fig. 4B) and reversed by the addition of the
PKC-II-specific peptide. Fig. 5A shows that contraction of permeabilized ESO cells was inhibited by antibodies raised against PKC- and not by antibodies raised against the PKC-II, -, or - isozyme (ANOVA, p < 0.01).
Inhibition of DAG-induced contraction PKC- was concentration
dependent and reversed by the addition of the PKC--specific peptide
(Fig. 5B). These data support the translocation experiments and suggest
that PKC-II may mediate contraction of LES, whereas PKC- may
mediate contraction of ESO.
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Different N-myristoylated pseudosubstrate peptides
inhibit DAG-induced contraction of LES and ESO.
To characterize
further the specific PKC isozymes that mediate contraction of these two
different smooth muscle types, we used N-myristoylated
peptides derived from the pseudosubstrate sequences of PKC-,
-, -, and - (myr-PKC-, myr-PKC-, myr-PKC-, and
myr-PK) (Table 1) and examined their effect on DAG-induced
contraction of intact LES and ESO smooth muscle cells.
(myr-PKC-) and was not inhibited by myr-PKC-, myr-PKC-, or myr-PKC- (Fig. 6A). In contrast, DAG-induced
contraction of ESO was inhibited by myr-PKC- and was not inhibited
by myr-PKC-, myr-PKC-, or myr-PKC-. PKI had no effect on
either tissue type (Fig. 6B). These data support the hypothesis that
PKC- mediates contraction of LES, whereas PKC- mediates
contraction of ESO.
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in the LES and by myr-PKC- in the
ESO. The half-maximal response, calculated by logit transformation, was
seen at 3 × 1010 M myr-PKC- for ESO
and 4 × 1010 M myr-PKC- for
LES.
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| |
Discussion |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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PKC plays an important role in the signal transduction pathway mediating smooth muscle contraction (15-21). We have previously described PKC-dependent contraction in the circular smooth muscle of both the LES and ESO. In the LES, we found that in vivo tone is reduced by the PKC antagonists H7 and calphostin C but not by the calmodulin antagonist W7. We have demonstrated that LES tone is associated with the spontaneous activity of phosphatidylinositol-specific phospholipase C, resulting in submaximal production of Ins(1,4,5)P3 and DAG, which act synergistically to activate PKC in a calcium-dependent manner (3). Furthermore, we have shown that this PKC-mediated contraction is augmented by additional DAG production arising from hydrolysis of phosphatidylcholine by phosphatidylcholine-specific phospholipases C and D (19).
In the ESO, we have shown that ACh-induced contraction requires the influx of extracellular calcium and activation of PKC and is independent of calmodulin because it is inhibited by PKC inhibitors such as H7, calphostin C, and chelerythrine and not affected by calmodulin inhibitors (1, 2). ESO contraction requires activation of phosphatidylcholine-specific phospholipase C (20), phospholipase D, and phospholipase A2 (21) and production of DAG and arachidonic acid without an associated increase in Ins(1,4,5)P3 (2, 8). In addition, we have shown that DAG-induced contraction of ESO is independent of cytosolic calcium, suggesting that the calcium influx is required primarily for the activation of the phospholipases responsible for the production of DAG and not for activation of PKC (2).
The results of the current study suggest that although both contractile
processes are PKC-dependent, different PKC isozymes (one
calcium-dependent and one calcium-independent) may mediate contraction
in these two distinct tissue types. Calcium-dependent PKC isozymes
(, I, II, and ) have a calcium-binding domain that allows
calcium and DAG to act synergistically on these PKC isozymes, whereas
calcium-independent isozymes (, , , , and ) lack this
calcium-binding domain and thus do not require calcium for activation
(22-24). In the current study, we showed that contraction of the
circular smooth muscle of the LES is mediated by the calcium-dependent PKC-II isozyme, whereas contraction of the circular smooth muscle of
the ESO is mediated by the calcium-independent PKC- isozyme. These
conclusions arise from the following findings.
Expression of PKC isozymes in LES and ESO smooth muscle.
Using
isoenzyme-specific antibodies in Western blotting experiments, we
demonstrate the presence of PKC-, -II, and - isozymes in the
circular smooth muscle layer of LES and of PKC-II, -, and -
isozymes in the ESO. These results are consistent with numerous studies
that report the expression of multiple PKC isozymes in smooth muscle.
Multiple PKC isozymes have been reported in rat aorta (25, 26), ferret
aorta (27), human saphenous vein and renal artery (28), hamster vas
deferens (28), and rat mesenteric artery (29). The functional
significance of the individual PKC isozymes expressed in the LES and
ESO was examined in our translocation and contractility experiments.
ACh stimulation causes PKC-II translocation in LES and PKC-
translocation in ESO.
In the current study, we demonstrated by two
distinct techniques that in LES, the calcium-dependent PKC-II
translocates from the cytosol to the membrane in response to ACh,
whereas in ESO, the calcium-independent PKC- translocates in
response to ACh.
and PKC- isozymes were identified in ferret aorta and shown
to translocate on agonist stimulation (27). It has been proposed that
translocation of PKC reflects PKC binding to intracellular receptors in
the particulate fraction (RACKs) and that binding to RACKs may be
required for PKC-mediated function. PKC binding to RACKs is specific,
dose dependent, and saturable and may confer specificity of isozyme
action by differential localization of isozyme-specific RACKs (30, 31).
We used Western blotting to examine the relative proportion of isozymes
in the cytosol and membrane before and after ACh stimulation. It is
notable that despite the presence of several distinct PKC isozymes in
LES, the membrane-to-cytosol density ratio changed only for PKC-II
(from 0.42 ± 0.15 to 2.44 ± 0.39). Similarly, in ESO, the
ratio changed only for PKC- (from 0.43 ± 0.14 to 2.61 ± 0.9).
These data are confirmed by activity measurements, which show that the
change in activity ratios of PKC-II in the LES and of PKC- in the
ESO is similar to the change in Western blot measurements of density
ratio.
We conclude that the translocating PKC-II in the LES and PKC- in
the ESO are the isozymes that may mediate contraction in these two
tissue types, possibly by bringing the enzymes to their specific RACKs
or membrane-bound effector proteins. The function of the other isozymes
is not presently known; they may participate in other cellular
functions.
DAG-induced contraction of LES and ESO cells is inhibited by
isozyme-specific PKC antibodies and isozyme-specific
pseudosubstrate-derived peptides.
We found that DAG-induced
contraction of permeabilized smooth muscle cells was dose-dependently
inhibited by antibodies raised against PKC-II in the LES and PKC-
in the ESO. In the LES, a complete concentration-response curve was
generated only for PKC-II because it was the only isozyme tested
that significantly inhibited contraction in the LES. Similarly, in ESO,
a complete concentration-response curve was generated only for PKC-
because it was the only isozyme tested that significantly inhibited
contraction in the ESO. In general, in this and other experiments using
antibodies, we found a maximal effect at a 1:200 concentration.
,
, , and µ, lack the C2 region, which has been
implicated in the regulation of PKC by calcium (32-34).
Antibodies were raised against synthetic peptides containing sequences
unique to specific PKCs. PKC- and PKC- antibodies were derived
from peptide sequences from the variable V3 region, and
PKC-II and PKC- antibodies were derived from peptide sequences from the V5 region. The V5 region of the PKC
molecule is located at the carboxyl terminus on the catalytic domain,
adjacent to the substrate binding site. It is possible that the binding
of anti-PKC-II and anti-PKC- to peptide sequences in the
V5 region induces a conformational change in the molecule
that inhibits substrate binding. Alternatively, antibody binding may
inhibit PKC activity by inhibiting the phosphorylation of the kinase
itself. There exists some evidence of phosphorylation of PKC in the
V1, V2, and V5 regions, which is
required for PKC catalytic activity (35-37). Most likely, however, the
precise site of binding of the antibody to a specific PKC is not
absolutely important because immunoglobulins are much larger molecules
(180-500 kDa) than PKC (80-90 kDa) and may affect the structural
conformation of the enzyme, regardless of their specific binding site.
These data support the translocation studies and suggest that PKC-II
mediates contraction in the LES and that PKC- mediates contraction
in the ESO. This view is further supported by the finding that
isozyme-specific pseudosubstrate-derived peptides inhibit DAG-induced
contraction in the LES and ESO. All PKC isozymes contain an
autoinhibitory sequence, near the C1 domain, called the
pseudosubstrate domain that is thought to interact with the catalytic
domain to keep the enzyme inactive in resting cells. Allosteric
activators, such as DAG or phorbol esters, relieve this intramolecular
control by inducing a conformational change in the molecule that
liberates the substrate binding domain from the pseudosubstrate,
thereby activating the enzyme (38). Synthetic peptides based on the
pseudosubstrate sequences of individual isozymes might be specific
inhibitors because they exploit the substrate specificity of the enzyme
without interfering with ATP binding. A recent approach uses
modification of peptides by myristoylation to overcome the permeability
barrier of the plasma membrane. Myristoylated peptides that are based
on the pseudosubstrate sequence of PKC- and PKC- have been
reported to inhibit the PKC-mediated phosphorylation of the
myristoylated alanine-rich C kinase substrate protein (MARCKS) and
phospholipase D activation of human fibroblasts (39). We have
previously shown that preincubation of lacrimal gland acini for 60 min
with 107 M myr-PKC- results in 80%
inhibition of protein secretion by phorbol ester (9). In a recent
study, we reported that synthetic myristoylated peptides derived from
the pseudosubstrate sequences of PKC-, -, and -, three
isoforms that are present in lacrimal gland acini, inhibit phorbol
ester- and cholinergic agonist-induced protein secretion in a
concentration-dependent manner. We showed that these peptides neither
interfered with cholinergic-induced changes in cytosolic
Ca2+ concentration nor inhibited vasoactive intestinal
peptide-induced protein secretion, a response mediated by cAMP and
protein kinase A. Although these peptides did not show clear
selectivity when tested in vitro with purified recombinant
PKCs, we showed that they differentially affected phorbol ester- and
agonist-induced protein secretion in the lacrimal gland. We suggested
that in vivo, these peptides might be inhibiting (competing
for) the binding of their respective PKC isoform to crucial
intracellular receptors such as RACKs rather than inhibiting PKC
activity.
In the current study, we demonstrated isozyme-specific inhibition of
DAG-induced contraction of intact smooth muscle cells from the LES and
ESO. DAG-induced contraction of LES cells was significantly inhibited
by the myristoylated peptide corresponding to the pseudosubstrate
sequence of PKC- (myr-PKC-); whereas ESO contraction
was significantly inhibited by myr-PKC-. It is worth noting that
myr-PKC- differs from myr-PKC- by the addition to its amino
terminus of only five amino acids (Table 1). This modification seems to
make myr-PKC- unable to inhibit PKC- and thus makes the peptide
selective to the PKC- isozyme. In addition, the myristoylated
peptide derived from the sequence of the endogenous inhibitor of
cAMP-dependent protein kinase A, PKI, was used as a control for the
sequence specificity of the inhibitory effect. PKI had no effect on
DAG-induced contraction of either LES or ESO smooth muscle.
To conclude, PKC-dependent contraction of the circular smooth muscle of
the LES is mediated by the calcium-dependent PKC-II isozyme, whereas
contraction of the circular smooth muscle of the ESO is mediated by the
calcium-independent PKC- isozyme. Other PKC isozymes present in ESO
and LES may mediate other functions.
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Footnotes |
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Received August 19, 1996; Accepted November 11, 1996
Presented in part at the 9th International Conference of Second Messengers and Phosphoproteins, Nashville, Tennessee, 1995, and at the 1996 AGA meeting [Gastroenterology 110:A761 (1996)].
This work was supported by National Institutes of Health Grants DK28614 and EY06177 and KOSEF hacsim 961-0704-042-2.
Send reprint requests to: P. Biancani, Ph.D., G. I. Motility Research Lab., SWP5, Rhode Island Hospital & Brown University, 593 Eddy Street, Providence RI 02903. E-mail: piero_biancani{at}brown.edu
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Abbreviations |
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ESO, esophagus;
ACh, acetylcholine;
ANOVA, analysis of variance;
DAG, dioctanoylglycerol;
Ins(1, 4,5)P3, inositol-1,4,5-trisphosphate;
LES, lower
esophageal sphincter;
NC, nitrocellulose;
PKC, protein kinase C;
PKI, protein kinase I;
RACK, receptor for activated C kinase;
SDS, sodium
dodecyl sulfate;
PAGE, polyacrylamide gel electrophoresis;
EGTA, ethylene glycol bis(-aminoethyl
ether)-N,N,N
,N-tetraacetic
acid;
HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;
PKA, protein kinase A.
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References |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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