Department of Pediatrics, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0656
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ABSTRACT |
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We have investigated the hypothesis that
different contractile agonists activate distinct catalytic subunits of
phosphoinositide (PI) 3-kinase in smooth muscle cells. Endothelin
(107 M) induced a sustained
increase in PI 3-kinase activity at both 30 s and 4 min of stimulation
(151.5 ± 8.5% at 30 s and 175.8 ± 8.7% at 4 min,
P < 0.005). Preincubation of smooth muscle cells with the
tyrosine kinase inhibitor genistein (3 µM) resulted in a significant
inhibition of both C2
ceramide-induced and endothelin-induced PI 3-kinase activation and
contraction. Preincubation with herbimycin A, an Src kinase inhibitor
(3 µM), inhibited only C2
ceramide-induced PI 3-kinase activation and contraction. Western
blotting using Src kinase antibody showed that
C2 ceramide, not endothelin,
stimulated the phosphorylation of Src kinase. Western blotting and
immunoprecipitation with PI 3-kinase antibodies to the regulatory
subunit p85 and the catalytic subunits p110
and p110
indicated
that both endothelin and C2
ceramide interacted with the regulatory subunit p85; endothelin interacted with the catalytic subunits p110
and p110
, whereas C2 ceramide interacted only with
the catalytic subunit p110
. In summary,
C2 ceramide activated PI 3-kinase
p110
subunit by a tyrosine kinase-mediated pathway, whereas
endothelin-induced contraction, unlike
C2 ceramide, was not mediated by
the activation of Src kinase but was mediated by G protein activation
of both p110
and p110
subunits (type IA and IB) of PI
3-kinase.
Src kinase; G proteins; tyrosine kinase
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INTRODUCTION |
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PHOSPHOINOSITIDE (PI) 3-kinase is a cytosolic enzyme that plays key roles in mediating signaling, including receptor-stimulated mitogenesis, oxidative burst, membrane ruffling, and glucose uptake (17). The activation of PI 3-kinase results in an increase in cellular levels of D-3 phosphorylated phosphoinositides, such as phosphatidylinositol 3-phosphate (PI-3-P), phosphatidylinositol 3,4-bisphosphate, and phosphatidylinositol 3,4,5-trisphosphate, which have been proposed to act as second messengers (11, 14, 27).
PI 3-kinase is activated by a variety of growth factors, oncoproteins, and other cellular activators (3). Most of the factors that activate this enzyme do so by turning on a cellular protein tyrosine kinase. PI 3-kinase is activated by platelet-derived growth factor, colony-stimulating factor-1, insulin, hepatocyte growth factor, epidermal growth factor, and interleukin-2 (15). The receptors of all these factors either have intrinsic protein tyrosine kinase activity or activate associated tyrosine kinase of the Src family. In most of these cases, the activation of PI 3-kinase correlates with the recruitment of this enzyme from the cytosolic fraction to an activated protein tyrosine kinase (3). Thrombin and formyl peptides appear to activate PI 3-kinase via G protein-linked pathways (8, 28). In our current study, two contractile agonists (endothelin and C2 ceramide) were found to activate PI 3-kinase via different pathways, leading to contraction of colonic smooth muscle cells.
The heterodimeric form of PI 3-kinase is made up of a regulatory
subunit, p85, and a catalytic subunit, p110. At least two types of PI
3-kinase, in terms of the mode of activation, have been described in
mammalian cells. They are type IA, which is stimulated by
membrane-bound receptors activating tyrosine kinase, and type IB, which
is under direct control of the heterotrimeric GTP-binding proteins
(18). They have been structurally characterized as a heterodimer
consisting of 110-kDa catalytic subunits (p110, p110
) and 85-kDa
regulatory subunits (p85
, p85
; see Ref. 30). Stimulation of
tyrosine kinase receptors by extracellular signals phosphorylates
specific tyrosine residues located in the YMX M motifs of their own
receptors or adaptor molecules, such as insulin receptor substrate-1.
These phosphorylated proteins bind to the SH2 domains of p85 and
stimulate the lipid kinase activity (18). Type IB PI 3-kinase is
activated by
- and
-subunits of G proteins; the adaptor is
unknown, and the catalytic subunit p110
may or may not be associated
with the p85 adaptor (14). Recently, it was found that p110
was
associated with a noncatalytic p101 subunit (20). Several lines of
evidence indicate that G protein stimulates p110
in the absence of
p101 both in vitro and in vivo (9, 10, 12, 22, 24). G
are thought
to be the dominant physiological stimulants, whereas G
subunits of
the Gi but not Gq or G12
subfamilies only moderately activate p110
(11).
Our previous study on C2 ceramide
(an intracellular product of sphingomyelin hydrolysis; see Ref. 6) set
up a model showing that tyrosine kinase and Src kinase are upstream of
PI 3-kinase, leading to smooth muscle contraction. We have used
endothelin-1 (a potent vasoconstrictor) and
C2 ceramide (a contractile
agonist) on colon smooth muscle cells and found that
C2 ceramide activated the PI
3-kinase p110 subunit via activation of tyrosine kinase and
activation of Src kinase pathways, whereas endothelin activated the PI 3-kinase p110
subunit mainly through a different
pathway, most likely through the G protein-mediated pathway
(2, 11). C2 ceramide activated
type IA PI 3-kinase, and endothelin activated both type IA and type IB
PI 3-kinase.
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MATERIALS AND METHODS |
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Materials
The following reagents were purchased. Collagenase type II was purchased from Worthington Biochemical (Freehold, NJ); DMEM and genistein were from Life Technologies (Gaithersburg, MD); endothelin-1 was from Peninsula Laboratories (Belmont, CA); C2 ceramide was from Matreya (Pleasant Gap, PA); G protein-Sepharose was from Pharmacia Biotech (Piscataway, NJ); protein assay standard and goat anti-mouse IgG (heavy and light chains) horseradish peroxidase conjugate were from Bio-Rad (Hercules, CA); phosphotyrosine-specific antibody, PI 3-kinase p110Methods
Isolation of smooth muscle cells from rabbit rectosigmoid. New Zealand White rabbits were killed, and their internal anal sphincter consisting of the distal most 3 mm of the circular muscle layer and ending at the junction of skin and mucosa were removed by sharp dissection. A 5-cm length of the rectosigmoid orad to the junction was dissected and digested to yield isolated smooth muscle cells. Cells were isolated as previously described (2). The tissue was incubated for two successive 60-min periods at 31°C in 15 ml of HEPES buffer containing 0.1% collagenase (150 U/mg; Worthington CLS type II) and 0.01% soybean trypsin inhibitor. After the second enzymatic incubation period, the medium was filtered through 500 µm Nitex mesh. The partially digested tissue left on the filter was washed four times with 50 ml of collagenase-free buffer solution. Tissue was then transferred into 15 ml of collagenase-free buffer solution and was incubated for 30 min to allow the cells to disperse spontaneously. After a hemocytometric cell count, the cells were resuspended in collagenase-free HEPES buffer (pH 7.4). Each rectosigmoid yielded 10-20 × 106 cells.Agonist stimulation and immunoprecipitation of PI
3-kinase. About 5 × 106 cells in 500 µl of buffer
were treated with different agonists and inhibitors. For experiments
checking the effect of 0 Ca2+,
cells were incubated in collagenase-free buffer with 2 mM EGTA for 30 min before the addition of agonists. Cells were incubated with
C2 ceramide
(107 M) or endothelin
(10
7 M) for 30 s and 4 min,
after incubation with one of the antagonists [either genistein (3 µM) or herbimycin A (3 µM)] for 20 min. The reactions were
set at 37°C and were stopped by dry ice cooling. The precipitates
were collected and then washed three times, including centrifugation at
14,000 rpm for 10 s; the pellets were mixed with 1 ml of 1 mM sodium
orthovanadate in PBS (150 mM NaCl, 16 mM
Na2HPO4,
and 4 mM
NaH2PO4,
pH 7.4) and were vortexed for 10 s. At the end of the last washing, the
pellets were kept, and 400 µl of PI 3-kinase lysis buffer [50
mM
-glycerophosphate, 1.5 M EGTA, 1 mM sodium orthovanadate, 1 mM
dithiothreitol (DTT), 0.01 mg/ml aprotinin, 0.01 mg/ml leupeptin, and 1 mM phenylmethylsulonyl fluoride, pH 7.4] were added. Cells were
sonicated for 1 min and were centrifuged for 10 min at 14,000 rpm.
Supernatants were collected, and protein concentration was measured
using a Bio-Rad protein assay system. Protein concentrations were
adjusted to be 3-4 µg/µl with 300 µg proteins in each tube.
Anti-PI 3-kinase antibody (p85 subunit specific binding) was added (1.5 µl antibody/300 µg protein). After 1.5 h of rocking at 4°C, 50 µl of G protein-Sepharose were added, and samples were rocked at
4°C for another 2 h. Samples were then washed three times by
spinning at 14,000 rpm for 5 min followed by replacement of the
supernatant with 1 ml of 100 mM Tris-buffered saline (TBS) buffer (0.1 M Tris and 0.154 M NaCl, pH 7.5) and rocking for 5 min. At the end of
the last washing, the precipitates were resuspended, and 30 µl of PI
3-kinase buffer (20 nM Tris base, pH 7.4, and 10 nM
MgCl2) were added. The
precipitates were kept on ice before use.
PI 3-kinase activity determination.
The PI 3-kinase activity assay technique was based on previously
described techniques (10, 19, 31) with some modification. The details
were as follows: substrates were prepared by extracting the lipid
mixture of 10 µg of phosphatidylserine and 10 µg of
phosphatidylinositol in 1 ml of methanol-chloroform (1:1) followed by
sonication in 10 µl of PI 3-kinase buffer. The kinase reaction
included 15 µl of kinase buffer, 10 µl of lipid substrate mixture,
and 10 µl of kinase-containing beads. The reaction was set at
30°C for 10 min beginning by adding 10 µl of 20 µM
[-32P]ATP (2 µl
labeled ATP in 8 µl of 104 µM ATP buffer) with 20 µCi
radioactivity to phosphorylate the hydroxyl group of the substrate PI.
The product was PI-3-P.
Phospatidylserine worked as a lipid carrier to ease the reaction. Each
sample reaction was done in duplicate, and a negative control was
carried out by replacing the substrates with the same volume of PI
3-kinase. The reaction was stopped by adding 100 µl of 1 N HCl. The
lipid product was extracted in 160 µl of methanol-chloroform (1:1,
vol/vol). The product was resolved by TLC using
CHCl3-MeOH-NH4OH-dH2O
(45:35:1.5:8.5, vol/vol/vol/vol).
PI-3-P was used as standard, and 5 µl were loaded on the plate. Sample products were loaded at 40 µl
each. After the chromatographic development was finished, TLC plates
were dried and placed in a tank for 10 min filled with one spoon of solid iodine. The iodine worked as an indicator to show the position of
the product. Finally, the PI-3-P
region was densitometrically quantitated [units: optical density
(OD) · mm2] after autoradiography.
Data were analyzed on a Cricket Graph 1.2 statistical program using
ANOVA and analogous nonparametric tests (Student's
t-test, unpaired test, two-tailed
P value).
Immunoprecipitation with mouse monoclonal
phosphotyrosine antibody and Western immunoblotting using either a
monoclonal IgG anti-Src (pp60src) antibody, a mouse monoclonal PI
3-kinase p85 antibody, a polyclonal PI 3-kinase p110
antibody, or a polyclonal PI 3-kinase p110
antibody. Smooth muscle cells were counted on a
hemocytometer and were diluted with HEPES buffer as needed. Cells were
then treated with reagents for indicated periods. After treatment, the
cells were washed with PBS and then disrupted by sonication in lysis
buffer (20 mM Tris · HCl, 150 mM NaCl, 1 mM Na3VO4,
1 mM NaF, 1 mM
Na4M0O4,
1 mM DTT, 20 mM
Na2HPO4,
20 mM NaH2PO4,
20 mM
Na4P2O7 · 10H2O,
50 µg/ml DNase/RNase A, 10 µg/ml aprotinin, 10 µg/ml leupeptin,
10 µg/ml pepstatin A, 2 mM phenylmethylsulfonyl fluoride, 10 µg/ml
antipain, and 5 mM EDTA). Protein concentration was adjusted to
0.5-1 µg/µl, with the total protein amount at 400 µg.
Anti-phosphotyrosine antibody was added to each sample (20 µl/250
µg protein) and was rocked for 1.5 h followed by 2 h rocking after
addition of 40 µl G protein-Sepharose. Samples were centrifuged at
14,000 rpm. The precipitate (100 µg protein) was subjected to
SDS-PAGE (7.5% gel) and electrophoretically transferred to an
Immobilon P transfer membrane. The membrane was incubated with 5%
skimmed milk in TBS containing 0.1% Tween 20 (TBST) for 1 h at room
temperature and was washed with TBST. The filter was incubated with the
primary antibody (either anti-p85 or anti-Src at 1:500 and either
anti-p110
or anti-p110
at 1:200) in TBST-milk for 1 h at room
temperature. After being washed with TBST (two quick washings, 2 × 5 min then 2 × 15 min), the filter was incubated with
horseradish peroxidase anti-mouse IgG antibody at 1:5,000 in TBST-milk
for 1 h at room temperature. After being washed with TBS (without Tween
20), the enzymes on the filter were visualized with luminescent
substrates using an enhanced chemiluminescence kit.
Measurement of Src kinase activity.
Src kinase was measured by a radioenzyme assay using a Src kinase assay
kit (6). The assay system is based on phosphorylation of a specific
substrate peptide
[p34cdc2-(620)] by
the transfer of
[
-32P]ATP by Src
kinase. Aliquots (1.0 ml each) of the smooth muscle cell suspension (2 × 106 cells/ml) were
incubated with reagents from 15 s to 20 min at 37°C. The incubation
was stopped with 1 ml of chilled HEPES buffer. The suspensions were
immediately centrifuged at 10,000 rpm for 50 s at 4°C in a
microfuge, and the supernatant was removed. The resultant pellet was
resuspended in 30 µl of chilled HEPES buffer (pH 7.4) containing 50 mM
-glycerophosphate, 25 mM NaF, 1% Triton X-100, 150 mM NaCl, 20 mM EGTA, 15 mM MgCl2, 1 mM DTT, 25 µg/ml leupeptin, and 25 µg/ml aprotinin (extraction buffer). Each
suspension (20-30 samples) was immediately frozen in liquid
nitrogen and was stored at
70°C overnight. The suspensions
were thawed and sonicated for 30 s at 4°C. The sonicates were
vortexed, allowed to settle for 10 min at 4°C, and then centrifuged
at 10,000 rpm for 15 min at 4°C. Each 10-µl sample of the
supernatant (~30 µg protein) was combined with 20 µl of substrate
solution, which contained 10 µg Src substrate peptide
[p34cdc2-(6
20):
Lys-Val-Glu-Lys-Ile-Gly-Glu-Gly-Thr-Tyr-Gly-Val-Val-Tyr-Lys], 50 mM Tris · HCl, 62.5 mM
MgCl2, 12.5 mM
MnCl2, 1 mM EGTA, 0.125 mM sodium orthovanadate, and 1 mM DTT (assay dilution buffer, pH 7.2).
For the control reactions, 10 µl of the supernatant from corresponding cell extracts were combined with 20 µl of assay dilution buffer without Src substrate peptide. All procedures were
performed at 4°C. Aliquots (10 µl) of
[
-32P]ATP (2 µCi)
dissolved in 2× assay dilution buffer, 500 µM ATP, and 75 mM
MnCl2 were added to each sample at
30°C. After incubation for 10 min at 30°C, the reaction was
stopped with 20 µl of ice-cold 40% TCA. All samples were placed on
ice for 10 min and were centrifuged at 10,000 rpm for 10 min to
precipitate any extract proteins. Supernatant (25 µl from each tube;
total volume, 60 µl) was removed, spotted on separate p81
phosphocellulose papers, and mounted on a piece of aluminum foil. Each
disk was placed in a glass scintillation vial containing 15 ml of
0.75% phosphoric acid. After 30 min of mixing at room temperature, the
washing reagent was decanted. Each paper was mixed with 10 ml of 0.75%
phosphoric acid, followed by 10 ml acetone, for 10 min at room
temperature. Each reagent was decanted after each washing. Finally, 10 ml of scintillant were added, and the radioactivity remaining on each
binding paper was counted in a liquid scintillation counter.
Nonspecific binding of
[
-32P]ATP to the
binding paper without substrate was subtracted from each control sample
that included the substrate. Src kinase activities were expressed as
picomoles per minute per milligram protein. The protein in each sample
(same fractions as Src kinase measurements) was measured using a
Bio-Rad protein assay system.
Measurement of contraction. Aliquots consisting of 2.5 × 104 cells in 0.5 ml of medium were added to 0.1 ml of a solution containing the test agents. The agents were agonists or combinations of agonists and inhibitors. The reaction was interrupted by the addition of 0.1 ml of acrolein at a final concentration of 1% (vol/vol). Individual cell length was measured by computerized image micrometry. The average length of cells in the control state or after addition of test agents was obtained from 50 cells encountered randomly in successive microscopic fields. The contractile response is defined as the decrease in the average length of the 50 cells and is expressed as the absolute change or the percent change from control length (1).
Preparation of permeable smooth muscle cells and
preincubation with G
antibody. In experiments involving preincubation of smooth muscle cells with G
antibody, muscle cells were made permeable without affecting their overall function (1). The partially
digested muscle tissue was washed with 50 ml of a "cytosolic" enzyme-free medium (cytosolic buffer) of the following composition (in
mM): 20 NaCl, 100 KCl, 5.0 MgSO4,
0.96 NaH2PO4,
25.0 NaHCO3, 1.0 EGTA, and 0.48 CaCl2. The medium contained 2%
BSA and was equilibrated with 95%
O2-5%
CO2 to maintain a pH of 7.2. Muscle cells were allowed to disperse spontaneously in this medium and were harvested by filtration on 500 µm Nitex mesh. Isolated cells were permeabilized by incubation for 3 min in saponin (75 µg/ml). The
cell suspension was centrifuged down and was resuspended in the
cytosolic buffer containing antimycin A (10 µM), ATP (1.5 mM), and an
ATP-regenerating system consisting of creatine phosphate (5 µM) and
creatine phosphokinase (10 U/ml). The permeabilized cells were allowed
to rest in a 95% O2-5%
CO2 environment for 30 min. Five
million cells in 0.1 ml cytosolic buffer were incubated with 2 µl of
G
antibody for 1 h before the addition of agonists.
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RESULTS |
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C2 Ceramide and Endothelin Induced PI 3-kinase Activities, and the Activation was Ca2+ Dependent
Our previous studies have shown that both C2 ceramide and endothelin induced contraction of smooth muscle cells at 30 s and 4 min (2, 6, 16). Their effect on PI 3-kinase activity was studied in this paper. C2 ceramide, the permeable short-chain C2 ceramide (10
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C2 Ceramide and Endothelin Induce PI 3-Kinase Activities Via Different Pathways
We next investigated whether C2 ceramide and endothelin activated the same signal transduction pathway in the activation of PI 3-kinase. We have previously shown (6) that C2 ceramide-induced Src kinase activities are through a cascade whereby PI 3-kinase is downstream of Src kinase in the signal transduction cascade involving the protein tyrosine kinases (see model in Fig. 3). Herbimycin A (3 µM), an Src kinase inhibitor, was used in our study to see if it has any effect on agonist-induced PI 3-kinase activities.
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There was a significant inhibition of
C2 ceramide-induced PI 3-kinase
activities by herbimycin A at 30 s (101.3 ± 11.0%,
P < 0.05, n = 4) and at 4 min (63.8 ± 6.8%,
P < 0.05, n = 4; Fig. 4). The inhibitory effect of genistein (3 µM), a tyrosine kinase inhibitor, and herbimycin A (3 µM) was
tested on Western immunoblotting, showing lower-density bands in
herbimycin A- and genistein-pretreated cells than
C2 ceramide alone (Fig.
2A). Thus the data suggest that C2
ceramide-induced activation of PI 3-kinase was through an Src
kinase-related protein tyrosine kinase cascade and that PI 3-kinase was
downstream of Src kinase.
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There was a slight inhibition of endothelin-induced PI 3-kinase
activities by herbimycin A at 30 s (129.2 ± 4.7%,
P < 0.05, n = 4) and at 4 min (144.2 ± 13.2%, P < 0.05, n = 4; Fig.
5). Western immunoblotting supported these
results (Fig. 2B). The data suggest that
endothelin-induced activation of PI 3-kinase was not dependent on
Src kinase and that a tyrosine kinase was upstream of PI 3-kinase (see
model in Fig. 3). Western immunoblotting showed that preincubation with
genistein inhibited endothelin-induced PI 3-kinase activation
(Fig. 2B).
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The same results relative to PI 3-kinase activation by C2 ceramide and endothelin were found on Western immunoblotting using immunoprecipitates of PI 3kinase subunit p85 followed by immunoblotting with phosphorylated tyrosine kinase antibody (Fig. 2C).
Another experiment was done using Western immunoblotting (Fig.
6C) to
find out if C2 ceramide or
endothelin phosphorylated Src kinase in the protein tyrosine kinase-PI
3-kinase cascade. C2 ceramide, not
endothelin, phosphorylated Src kinase; an increase in the protein
phosphorylation corresponding to Src kinase (60 kDa) was notable in
response to C2 ceramide but not
endothelin. Src kinase activity assay showed that there is a
significant increase of Src kinase activity (units:
pmol · min1 · mg
protein
1) stimulated by
C2 ceramide at 30 s and 4 min
(3.47 and 4.13) compared with control (1.27), whereas endothelin showed
almost no activation of Src kinase at 30 s and 4 min (1.26 and 1.27). Thus results from Western blotting of phosphorylated Src kinase and
from measurement of Src kinase activity confirmed that
endothelin-induced activation of PI 3-kinase is Src kinase independent.
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C2 Ceramide and Endothelin Activate Different Types of PI 3-Kinase
We investigated whether C2 ceramide or endothelin interacted with different catalytic subunits. At least two groups of PI 3-kinase have been identified (18), namely, type IA with p85 as regulatory site and p110
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Endothelin-Induced PI 3-Kinase Activation Is Through G Protein
-Subunit
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Smooth Muscle Contraction Induced by C2 Ceramide and Endothelin Responded Differently to the Src Kinase Inhibitor Herbimycin A
Our previous study found that both C2 ceramide and endothelin induced contraction in colonic smooth muscle cells (1, 6). In this study, cells were preincubated with either the tyrosine kinase inhibitor genistein (3 µM) or the Src kinase inhibitor herbimycin A (3 µM) for 20 min before the addition of either C2 ceramide (10 ![]() |
DISCUSSION |
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We have investigated the different signal transduction mechanisms of contraction induced by endothelin and C2 ceramide, specifically in their respective activation of PI 3-kinase. Both C2 ceramide and endothelin induce contraction of smooth muscle cells isolated from the rabbit colon (1, 6). Endothelin is a potent vasoconstrictor. Its physiological importance in the control of gastrointestinal motor function has been investigated (2). Endothelin receptors are present on smooth muscle cells in jejunum, ileum, and colon (7, 23). C2 ceramide is the product of hydrolysis of sphingomyelin and acts as an intracellular messenger that mediates sustained smooth muscle contraction (16).
We have previously shown on Western immunoblotting that C2 ceramide-induced contraction is mediated by an increase in PI 3-kinase (32). We have further shown that, in C2 ceramide-induced contraction, PI 3-kinase is downstream of Src kinase in the protein tyrosine kinase cascade. Our data indicate that C2 ceramide increased PI 3-kinase activities at both 30 s and 4 min after stimulation with C2 ceramide. This increase in PI 3-kinase activities was inhibited by preincubation of the cells with genistein (a tyrosine kinase inhibitor) or preincubation with herbimycin A (an Src kinase inhibitor; Figs. 2A and 4). There was a close correlation between the Ca2+ requirement for C2 ceramide-induced contraction and C2 ceramide-induced PI 3-kinase activity. The data indicate that the sustained PI 3-kinase activity in response to C2 ceramide (4 min) is dependent on Ca2+ from extracellular sources (Fig. 1). Extracellular Ca2+ is also required to maintain sustained contraction induced by C2 ceramide at 4 min (6).
Similar results as above were found in response to stimulation by endothelin. It has been reported that, in vascular smooth muscle cells, endothelin acts either to mobilize intracellular Ca2+ or to promote Ca2+ influx (13, 33). Our results indicate that, in smooth muscle cells from the rabbit colon incubated in buffer lacking extracellular Ca2+ (0 Ca2+-2 mM EGTA), endothelin-induced sustained contraction is inhibited at 4 min (2). The contractile response to endothelin seems to be in accord with endothelin-induced PI 3-kinase activity. PI 3-kinase activity in response to endothelin is greatly inhibited at 4 min stimulation in the presence of 0 Ca2+-2 mM EGTA (Figs. 1B and 2B).
Endothelin seems to activate PI 3-kinase in a G protein-mediated
pathway, independent of the Src kinase pathway. The data indicate that,
on Western immunoblotting (Fig. 6C),
endothelin-induced contraction was not accompanied by any Src kinase
phosphorylation. Furthermore, endothelin did not induce any increase in
Src kinase activity in association with an increase in PI 3-kinase
activation (Fig. 7). Endothelin-induced PI 3-kinase activation is
through the G protein -subunit, which corresponds to the finding
on Western blotting (Fig. 8), whereby preincubation with the G protein
-subunit antibody blocked the endothelin-induced PI 3-kinase activation. We have previously shown (2) that specific G proteins mediated endothelin-induced contraction. The transmembrane signaling of
endothelin is through two specific GTP-binding components that are
Gi
; a
Gi-3-like protein appeared to be
required for the initial rapid transient contraction, and a
Gi-1-like protein is required for
the sustained phase of the contraction. It was recently reported that
the catalytic subunit of PI 3-kinase (p110
) was greatly stimulated
by G
(11). It was also suggested that G
can bind directly
to p110
(10). Thus it is possible for an agonist like endothelin,
which acts through a G protein-mediated response (2), to activate
G
and thus interact with either or both p110
and p110
catalytic subunits. Therefore, endothelin most likely activates the PI
3-kinase catalytic subunit independent of Src kinase.
It was recently found that serine kinase is tightly associated with PI 3-kinase and acts as a negative regulator of this enzyme; the best substrate found is the p85 subunit of PI 3-kinase (4). When PI 3-kinase p85 subunit immunoprecipitates were immunoblotted using serine phosphorylated antibody, no phosphorylated serine signals at the position of 85 kDa were found in response to the stimulation by C2 ceramide and endothelin (data not shown). Therefore, serine kinase did not seem to be involved in the activation of PI 3-kinase induced by C2 ceramide and endothelin in rabbit colonic smooth muscle cells.
Type IA PI 3-kinase enzymes purify as heterodimers with a molecular
mass of ~200 kDa containing a catalytic subunit of 110 kDa (p110) and
a regulatory subunit p85 (10, 25, 26). Several mechanisms for
regulating their enzymatic activities in response to extracellular
stimuli have been elucidated. Type IA members p110, -
, and -
are stimulated by tyrosine phosphorylated proteins through interaction
with regulatory PI 3-kinase subunits such as p85 or p55 (5, 30). They
in turn bind to the NH2 terminus of the catalytic p110 subunit, thereby inducing PI 3-kinase activity. In contrast, type IB members have the catalytic subunit p110
(21).
Our results indicate that C2
ceramide interacted only with type IA PI 3-kinase and through the
tyrosine kinase cascade pathway, whereas endothelin interacted with
both IA and IB types of PI 3-kinase. The involvement of type IB PI
3-kinase in endothelin activation fits the hypothesis that endothelin
interacts with G protein in the cell membrane, activates PI 3-kinase
without activating Src kinase, and induces smooth muscle contraction. Type IA was reported to be regulated by tyrosine kinase and Ras (29).
We hypothesize that smooth muscle cells of the rabbit colon have
different types of PI 3-kinase that could be activated differentially
and specifically by different contractile agonists. Our data suggest
that a direct activation of the Src kinase pathway results in the
interaction with only type IA, whereas endothelin activates type IA and
IB PI 3-kinase, probably through activation of a tyrosine kinase first
followed by the binding of the p85 regulatory subunit to the
NH2 terminus of the p110
or
p110
catalytic subunit. Activation of both classes IA and IB by
endothelin is mediated by G proteins.
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ACKNOWLEDGEMENTS |
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We thank Lyla Melkerson Watson and Ulrika Aronsson for technical assistance. We also thank Erin McDaid-Kelly for the preparation of this manuscript.
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FOOTNOTES |
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This study was funded by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-42876.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other corrspondence: K. N. Bitar, Univ. of Michigan Medical School, 1150 W. Medical Center Dr., A520D, MSRB I, Ann Arbor, MI 48109-0656 (E-mail: bitar{at}umich.edu).
Received 17 April 1998; accepted in final form 5 December 1998.
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REFERENCES |
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Bitar, K. N.,
C. Hillemeier,
P. Biancani,
and
K. J. Balazovich.
Regulation of smooth muscle contraction in rabbit internal anal sphincter by protein kinase C and Ins(1,4,5)P3.
Am. J. Physiol.
260 (Gastrointest. Liver Physiol. 23):
G537-G542,
1991
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Bitar, K. N.,
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